Pharmaceutical agents, compositions, and methods relating thereto

ABSTRACT

The present disclosure provides compounds of formulas (1)-(3), and compositions and methods of use thereof. The present disclosure also provides methods of preparing a provided compound and composition, and methods of characterizing a provided compound and composition.

PRIORITY CLAIM

This application is a U.S. National Stage Application under 35 U.S.C.371 of International Patent Application No. PCT/US18/30371, filed May 1,2018, which claims priority to U.S. Provisional Patent Application Ser.No. 62/508,730, filed May 19, 2017. The entire content of eachapplication is incorporated herein by reference.

BACKGROUND

Diabetes is a group of metabolic diseases in which there are high bloodsugar levels over a prolonged period. There are various types ofdiabetes. Type I diabetes results from the pancreas's failure to produceenough insulin. Type II diabetes begins with insulin resistance, acondition in which cells fail to respond to insulin properly, and maycause a lack of insulin as the disease progresses. There are about 400million diabetes patients worldwide, with type II diabetes making upabout 90% of the cases. Insulin, or insulin analogs, are generally usedfor treating type I diabetes. Metformin is generally recommended as afirst line treatment for type II diabetes.

SUMMARY

Prior work (see WO2015/137983, US2016/0045533 (issued at U.S. Pat. No.9,642,874), and US2016/0082033, each of which is incorporated herein byreference) has defined certain selenoorganic compounds that showinteresting and valuable activity in certain disease model systems.Specifically, this work has defined compounds of formulas I, II, andIII, depicted below:

for which it reports that:

-   -   1. A combination of the following three compounds:        5′-Methylselenoadenosine (compound C, which is a compound of        formula I), Se-Adenosyl-L-homocysteine (compound D, which is a        compound of formula II), and        Gamma-glutamyl-methylseleno-cysteine (Compound E, which is a        compound of formula III),

 but not the individual compounds, can significantly attenuate G6pcexpression, thereby representing a novel way to reduce hepatic glucoseoutput (See, WO2015/137983);

2. Compounds C and D each individually can enhance mitochondrial (MT)potential in mouse skeletal muscle myoblast C2C12 cells, which suggeststhat compounds C and D can be potentially useful in the area of T2DMresearch and control (See, WO2015/137983);

3. A combination of compounds C, D and E can reduce hepatic glucoseoutput and improve glucose tolerance in an insulin-resistant, diabeticmouse model, so that a combination of compounds C, D and E can be usefulin treatment of obesity, hyperglycemia, and diabetes (See,US20160045533);

4. Compounds C and D may be useful in treating sarcopenia caused byprogressive loss of MT function in the kidney or skeletal muscle (See,WO2015/137983);

5. Compound C can enhance gluconeogenesis in brain cells which may bebeneficial for the survival of brain cells in AD (See, US20160082033);and

6. Compounds C and D can inhibit Tau hyperphosphorylation in AD brains(See, US20160082033).

Thus, prior work demonstrates that compounds C, D, and E may be usefulin certain contexts; specifically teaching that, in some contexts (e.g.,inhibiting Tau hyperphosphorylation in AD brains, and enhancinggluconeogenesis in brain cells), compound C or compound D might beuseful alone. In other contexts (e.g., reducing hepatic glucose output,improving glucose tolerance and/or otherwise effectively treatingobesity, hyperglycemia, and/or diabetes), these compounds are shown notto be useful individually, but to be effective in combination.

The present disclosure provides new selenium-containing compounds,sharing some structural relationship with compounds of formulas I and IIabove (and specifically with compounds C and D), that surprisingly showpotent activity alone in a variety of contexts.

The present disclosure demonstrates, among other things, that providedcompounds exhibit bioactivity comparable to or better than the CDEcombination in reducing hepatic glucose output and/or improving glucosetolerance in insulin-resistant, diabetic subjects. The presentdisclosure also teaches that provided compounds likely have bioactivitycomparable to or better than Compounds C and/or D in enhancinggluconeogenesis (e.g., in brain cells), and/or in inhibiting Tauhyperphosphorylation (e.g., in AD brains).

The present disclosure provides compositions that contain and/or deliversuch compounds (and/or one or more degradants and/or active metabolitesthereof), as well as various methods (e.g., of manufacture,characterization, and/or use) and/or materials (e.g., intermediates,degradants, metabolites [in particular, active metabolites], etc)related to such provided compounds. In some embodiments, providedtechnologies relate to and/or are particularly useful in modulatingglucose metabolism; enhancing AS160 phosphorylation for translocation ofglucose transporter proteins (GLUTs) from cytosolic vesicles to plasmamembrane for glucose uptake; and/or enhancing glucose uptake in bothliver and skeletal muscles. In some embodiments, provided technologiesrelate to and/or are particularly useful in treatment ofhyperinsulinemia, obesity, diabetes, hyperglycemia, polycystic ovarysyndrome (PCOS), Alzheimer's disease (AD), and/or sarcopenia. In someembodiments, provided technologies relate to and/or are particularlyuseful in treatment of type II diabetes related disorders, such asdiabetic retinopathy, nephropathy, neuropathy, and vascular disorders.

In some embodiments, the present disclosure provides a compound offormula (1):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,wherein

each of R² and R³ is independently H or —C(O)—R, wherein each R isindependently C₁₋₆alkyl or 3-8 membered carbocyclic or heterocyclic,wherein R² and R³ cannot be both H;

or R² together with R³ form —(CH₂)_(n)—C(O)—(CH₂)_(m)—, wherein each ofn and m is independently 0-3, and n+m≤3;

R⁵ is —C₁₋₆alkyl or —C₁₋₆alkyl-CH(NH₂)COOH;

R⁸ is H or halogen; and

X is H or halogen,

wherein each of the carbocyclic, heterocyclic, —(CH₂)_(n)—, and—(CH₂)_(m)— moieties, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and

each C₁₋₆alkyl moiety, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, or CN.

In some embodiments, the present disclosure provides a compound offormula (2):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,

wherein R⁸ is H or halogen;

X is H or halogen;

each R₅′ is independently H or halogen; and

each R is independently C₁₋₆alkyl, each of which, independently, mayoptionally be substituted 1-3 times by halogen.

In some embodiments, the present disclosure provides a compound offormula (3):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,

wherein R⁸ is H or halogen;

X is H or halogen; and

each R′ is independently H or halogen.

In some embodiments, the present disclosure provides compositions whichcomprise or deliver a compound of any one of formulas (1)-(3). In someembodiments, the present disclosure provides compositions comprising acompound of any one of formulas (1)-(3), or a pharmaceuticallyacceptable salt, prodrug, or isomer thereof. In some embodiments, thepresent disclosure provides compositions which deliver an active moietyof a compound of any one of formulas (1)-(3).

In some embodiments, the present disclosure provides methods of treatinga disease, disorder, or condition by administering a compound orcomposition as described herein. In some embodiments, provided methodsenhance AS160 phosphorylation for translocation of glucose transporterproteins (GLUTs) from cytosolic vesicles to plasma membrane for glucoseuptake. In some embodiments, provided methods enhance glucose uptake inboth liver and skeletal muscles. In some embodiments, provided methodsattenuate hyperinsulinemia without impairing kidney function and/orresulting in liver damage.

In some embodiments, the present disclosure provides methods fortreating an insulin-related disorder, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods fortreating insulin resistance disorder comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods fortreating glucose metabolism disorders, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, glucose metabolism disorders involve ablood glucose level which is not within the normal range. In someembodiments, glucose metabolism disorders relate to defective glucoseuptake and/or transport. In some embodiments, glucose metabolismdisorders are Diabetes Mellitus, glyceraldehyde-3-phosphatedehydrogenase deficiency, glycosuria, hyperglycemia, hyperinsulinism, orhypoglycemia.

In some embodiments, the present disclosure provides methods fortreating disorders of glucose transport, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, disorders of glucose transport areglucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivodisease (GLUT1 deficiency syndrome (GLUT1DS)).

In some embodiments, the present disclosure provides methods fortreating obesity comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating diabetes comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating hyperglycemia comprising administering a therapeuticallyeffective amount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating polycystic ovary syndrome (PCOS) comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods fortreating Alzheimer's disease (AD) comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods fortreating sarcopenia comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forinhibiting glucose production, comprising administering a compound ofany one of formulas (1)-(3), or a pharmaceutically acceptable salt,prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forincreasing glucose tolerance, comprising administering a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods foractivating and/or restoring insulin receptor function and its downstreamsignaling in a subject in insulin-resistant state, comprisingadministering a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating mitochondria-associated diseases (e.g., caused by dysfunctionalmitochondria), comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, mitochondria-associated diseases can be degenerativediseases (e.g., cancer, cardiovascular disease and cardiac failure, type2 diabetes, Alzheimer's and Parkinson's diseases, fatty liver disease,cataracts, osteoporosis, muscle wasting such as sarcopenia, sleepdisorders and inflammatory diseases such as psoriasis, arthritis andcolitis). In some embodiments, the present disclosure provides methodsfor enhancing mitochondrial function, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods forenhancing gluconeogenesis in the brain, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, provided methods increase glucose uptakein the brain. In some embodiments, provided methods are for maintainingor restoring brain functions including memory and learning.

In some embodiments, the present disclosure provides methods forpreparing a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forcharacterizing a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forpreparing a composition as described herein.

In some embodiments, the present disclosure provides methods forcharacterizing a composition as described herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Effects of insulin and pure compounds (listed in Table 1) onglucose production in HepG2 cells. Cells were treated with 0.24% DMSO(the maximal volume of tested compound solvent), insulin or listedcompounds in serum-free glucose production media for 48 hr. Data werenormalized by cell number as described above, and presented as mean±SEMof at least 3 samples per group.

FIG. 2. Comparison of the potency of Compound #43 and metformin in bothHepG2 and H4IIE cells. HepG2 cells were treated with 0.24% DMSO (themaximal volume of Compound #43 solvent), insulin, Compound #43 andmetformin in serum-free glucose production media for 48 hr, while ratliver cells were treated for 24 hr. Glucose levels in culture media werenormalized by cell number in each sample. Data are presented as mean±SEMof between 3 and 8 samples per group.

FIG. 3. Differential effects of Compound #C, #50 and #43 on bloodglucose levels and serum HbA1c levels in Lepr^(db/db) mice after chronictreatment. Lepr^(db/db) mice were intraperitoneally injected with saline(containing 0.2% the compound solvent DMSO), Compound #C, #50 and #43 atthe dose of 25 μg selenium of each compound per kilogram body weightdaily starting 38 days of age. At the mouse age of day 81, the bloodglucose level of overnight-fasting mice were determined using aglucometer. Serum HbA1c levels were determined on mice at 90 days ofbirth. The relative HbA1c levels were obtained after the HbA1c levels inCompound #C-, #50- and #43-treated mice were divided by the averageHbA1c levels in saline-treated mice. Data are presented as mean±SEM ofindicated number of animals. P values were derived by comparingtreatments to the control/saline group.

FIG. 4. Effects of Compound #43 and its sulfur analog #68 on bloodglucose levels and HbA1c levels in Lepr^(db/db) mice after chronictreatment. Lepr^(db/db) mice were intraperitoneally injected with saline(containing 0.2% the compound solvent DMSO), Compound #43, and #68 at adose of 25 μg selenium or sulfur as Compound #43 (0.136 mg) or Compound#68 (0.298 mg), respectively, per kilogram body weight daily starting at38 days of age. At the mouse age of day 128, blood glucose and HbA1clevels of overnight-fasting mice were determined. HbA1c levels inCompound #43 and #68-treated mice were divided by the average HbA1clevels in saline-treated mice to obtain the relative HbA1c levels. Dataare presented as mean±SEM of the indicated number of animals. Fastingblood glucose levels in non-diabetic/obese mice (indicated by the symbol#in the bar graph) were obtained from 4-month-old wild-type C57 malemice in the laboratory. Different letters in the bar graph indicate a Pvalue less than 0.05.

FIG. 5. Differential effects of Compound #43, #69 and #70 on fastingblood glucose and HbA1c levels in Lepr^(db/db) mice after chronictreatment. Male 41-day-old Lepr^(db/db) mice were intraperitoneallyinjected with saline (containing 0.2% the compound solvent DMSO),Compound #43 (0.136 mg), #69 (0.145 mg) and #70 (0.153 mg) at the doseof 25 μg selenium of each compound per kilogram body weight daily for 43days (for blood glucose assay) and 90 days (for HbA1c assay), fastedovernight, and then subjected to blood glucose analysis (using aglucometer) or blood HbA1c assay. Data are presented as mean±SEM ofindicated number of animals. P values were derived by comparingtreatments to the control/saline group.

FIG. 6. Acute treatment of Compound #43 resulted in a decrease in bloodglucose level in Lepr^(db/db) male mice. 8-10-week-old Lepr^(db/db) malemice were fasted overnight, and then injected intraperitoneally withsaline (containing 0.2% DMSO, the maximal injected volume of Compound#43 stock solvent), 0.0054, 0.054, 0.54 and 5.4 mg Compound #43/kg bodyweight. Blood glucose levels in Lepr^(db/db) mice right before and afterinjection at 1, 2, 3, 5 and 8 hours (under fasting conditions but havingfree access to water) were examined. The reduced glucose levels inindividual mice were obtained by subtracting the glucose level rightbefore the injection from the blood glucose level at each time periodafter injection. Data are presented as mean±SEM of the indicated numberof animals. With the exception of the P values shown, which relate tothe early time points of the 0.0054 mg/kg BW treatment, all otherreductions were significant (P<0.05) when compared with thecorresponding time points for DMSO/saline injection.

FIG. 7. Acute treatment of Compound #43 reduced the blood glucose levelsin Lepr^(db/db) male mice under ad-libitum feeding conditions. Bloodglucose levels of 6-week-old Lepr^(db/db male) mice with free access tofood and water were determined before and at 24 hr after anintraperitoneal injection of saline (containing 0.2% the compoundsolvent DMSO) or Compound #43 at a dose of 5.4 mg/kg body weight. Therelative blood glucose levels before i. p. injection was normalized bythe average glucose level of all five mice within the group, andreferred to as 100%. After 24 hr injection, the relative blood glucoselevel in each mouse was normalized by its glucose level beforeinjection. Different letters represents a statistically significantdifference (P<0.05) between groups.

FIG. 8. Chronic treatment of Compound #43 improves glucose tolerance inLepr^(db/db) mice. A-B. Male 38-day-old Lepr^(db/db) mice wereintraperitoneally injected with (A) saline (containing 0.2% the compoundsolvent DMSO), Compound #43, Compound #C and Compound #50 daily for 43days, or with (B) Compound #68 or Compound #43 for 60 days. C. Male41-day-old Lepr^(db/db) mice were intraperitoneally injected with saline(containing 0.2% the compound solvent DMSO), Compound #43, Compound #69and Compound #70 daily for 43 days. The daily injected dose of alllisted compounds was 25 μg selenium or sulfur per tested compound perkilogram body weight. At the end of treatment, these mice were fastedovernight, injected with glucose (2 g/kg body weight) and blood glucoselevels immediately before glucose injection (referred to as zero timepoint) and at 0.25 hours, 0.5 hours, 1 hour and 2 hours post-glucoseinjection were measured using a glucometer with the maximal reading of600 mg/dL. A glucose level in excess of this limit was recorded as 600mg/dL. Data are presented as Mean±SEM of indicated number of animals.*P<0.05, **P<0.01, ***P<0.001 when compared to (A, C) saline-treated or(B) Compound #68-treated mice at the same time point.

FIG. 9. Attenuated G6pc mRNA in the livers of Lepr^(db/db) mice afterchronic treatment with Compound #43. Lepr^(db/db) mice at postnatal day38 were intraperitoneally injected with saline (containing 0.2% thecompound solvent DMSO), Compound #50 and #43 at the dose of 25 μgselenium of each compound per kilogram body weight daily for 52 days.QRT-PCR was performed on liver RNA isolated from these compound-treatedmice. G6pc mRNA level in each sample was normalized by Actb mRNA leveland data are presented as mean±SEM of five mice per group. P value isrelative to the saline group.

FIG. 10. Inhibition of basal G6pc/G6PC expression by Compound #43 inAML-12 and human HepG2 cells, and the cooperative action of bothCompound #43 and insulin in the inhibition of G6pc expression in AML-12cells. (A) Effects of selenium compounds on G6pc expression in AML-12cells under serum-free conditions. AML-12 cells were treated without(Control), or with compound CDE combination, Compound #C, Compound #D,Compound #50 and Compound #43 at a dose of 300 parts per billion (ppb)of selenium (equivalent to 3.8 uM of each compound) in serum-free,Insulin-Transferrin-Sodium selenite supplement (ITS) and Dexamethasone(Dex)-free media for 24 hr. (B) Inhibition of G6PC expression byCompound #43 in human HepG2 cells. HepG2 cells were incubated with 100nM of insulin or 600 ppb of Compound #43 in serum-free media for 40 hr.(C) Inhibition of G6pc expression by Compound #43, and the cooperativeaction of both Compound #43 and insulin in the inhibition of G6pcexpression in AML-12 cells that were pretreated with Compound #43.AML-12 cells were pretreated with Compound #43 in FBS-containing butITS/Dex-free media for 24 hours followed by retreatment of Compound #43in the presence or absence of 10 nM insulin in serum/ITS/Dex-free mediafor 6 hours. G6pc mRNA level in each sample was normalized by Actb mRNAlevel and data are presented as mean±SEM of indicated number of samplein each group. P value in panel A-B was compared to the Control group.Different letters in panel C represents a statistical significance(P<0.05) between those two groups.

FIG. 11. Inhibition of G6pc expression by Compound #43, and thecooperative action of both Compound #43 and insulin in the inhibition ofG6pc expression in AML-12 cells stimulated with diabetic stimuli. AML-12cells were pretreated with Compound #43 in 10% FBS-containing butITS/Dex-free media for 24 hours followed by retreatment of Compound #43in the presence or absence of insulin along with 8-CPT/Dex (diabeticstimuli) in serum-free and ITS-free media for 6 hours. G6pc mRNA levelin each sample was normalized by Actb mRNA level and data are presentedas mean±SEM of indicated number of sample in each group. Numbers on thetop of each column are the mean values of G6pc mRNA levels normalized bythe level in non-8-CPT/Dex-treated group (Column #1). Different lettersin the bars represents a statistical significance (P<0.05) between thosetwo groups.

FIG. 12. Chronic treatment of Compound #43 enhanced the phosphorylationof Pdk1/Akt/Foxo1 signaling in the livers of insulin-resistantLepr^(db/db) mice by Western blot analysis. Lepr^(db/db) mice atpostnatal day 38 were intraperitoneally injected with saline (containing0.2% the compound solvent DMSO) or Compound #43 at a dose of 25 μgselenium per kilogram body weight daily for 52 days. Western blots wereperformed on liver tissues (100 ug protein per lane) from saline orCompound #43-treated mice. (A) Western blot images. Protein levels ineach sample were normalized by Gapdh level and data are presented asmean±SEM of five mice per group in (B). P value was compared to thesaline group.

FIG. 13. Transient activation of PDK1 and AKT and subsequentinactivation of FOXO1 in human liver HepG2 cells by Compound #43. HepG2cells were serum-starved overnight, incubated without or with Compound#43 (600 ppb) for the indicated time, and then subjected to Western blotanalysis. (A) Representative Western blots. (B-F) Quantitative data ofprotein expressions of (B) pPDK1, (C) pAKT, (D) total AKT, (E)pFOXO1T24, and (F) total FOXO1 in Western blots are presented asmean±SEM of 3 samples. *P less than 0.05 when compared to the proteinlevel at 0 min (right before compound treatment) or its control group ateach time point.

FIG. 14. Transient activation of Pdk1 and Akt and enhanced Foxo1phosphorylation by Compound #43 in mouse liver AML-12 cells culturedunder simulated diabetic condition. AML-12 cells were cultured in 10%FBS but ITS/Dex-free DMEM/F12 media for 24 hr, and then serum-starved inplain DMEM/F12 media overnight. These serum-starved AML12 cells weretreated with diabetic stimuli, 8-CPT (0.1 mM) and Dex (0.5 μM), incombination without (Control) or with 10 nM insulin or Compound #43 (300ppb) in plain DMEM/F12 media for the indicated time points, and thensubjected to Western blot analysis. (A) Representative Western blots.(B-E) Quantitative data of protein expressions in Western blots arepresented as mean±SEM of 3 samples. *P<0.05 when compared to the Control(no insulin/Compound #43 treatment) at each time point. Differentletters in (E) represents a statistical significance (P<0.05) betweenthose two groups.

FIG. 15. Effects of Compound #43 and Compound #50 on the expression ofthe Glut4 gene in the livers of Lepr^(db/db) mice. Lepr^(db/db) mice atpostnatal day 38 were intraperitoneally injected with saline (containing0.2% the compound solvent DMSO), Compound #50 or Compound #43 at a doseof 25 μg selenium of each compound per kilogram body weight daily for 52days. QRT-PCR analyses were performed on liver RNA samples isolated fromthese compound-treated Lepr^(db/db) mice. Glut4 mRNA level in eachsample was normalized by Actb mRNA level, and data are presented asmean±SEM of four to five mice per group. P values were calculated fortreatment versus the control saline group.

FIG. 16. Enhanced Glut4 mRNA expression in mouse liver AML-12 cells byCompound #43. (A) QRT-PCR of basal Glut4 expression in AML-12 cells.AML-12 cells were amplified, seeded on 24-well plates, and cultured in10% FBS ITS/Dex-free DMEM/F12 media overnight. These cells were thenincubated with vehicle (0.024% DMSO) or Compound #43 (300 ppb) inserum-free DMEM/F12 media for 24 hours (hr). (B) QRT-PCR of Glut4expression in AML-12 cells cultured under simulated diabetic conditions.Amplified AML-12 cells were cultured on 24-well plates in 10% FBS butITS/Dex-free DMEM/F12 media for 24 hr, and then serum-starved in plainDMEM/F12 media overnight. Serum-starved AML-12 cells were then incubatedwith vehicle (0.024% DMSO) or with Compound #43 (300 ppb) in thepresence of diabetic stimuli, 0.1 mM 8-CPT and 0.5 μM Dex, in serum-freeplain DMEM/F12 media for 6 and 24 hr. Glut4 mRNA level in each samplewas normalized by Actb mRNA level, and data are presented as mean±SEM of3 samples.

FIG. 17. Enhanced glucose uptake in AML-12 cells after the treatment ofinsulin and Compound #43 for 1.5 hours. Data are presented as mean±SEMof the indicated number of samples per group. *P value was less than0.05, compared to the basal group.

FIG. 18. Enhanced phosphorylation of insulin downstream signalingmolecules-Pdk1, Akt and Foxo1—in skeletal muscles of insulin-resistantLepr^(db/db) mice in response to treatment with Compound #43.Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline (containing 0.2% the compound solvent DMSO) or Compound #43at the dose of 0.136 mg of Compound #43 per kilogram body weight dailyfor 52 days. Western blots were performed on skeletal muscle proteinextracts (100 μg protein per lane) isolated from saline or Compound#43-treated Lepr^(db/db) mice. (A) Images of Western blots. (B)Quantitative protein levels (normalized by β-tubulin protein levels ineach sample). Data are presented as mean±SEM of five mice. P value wasderived by comparison to the control (saline) group.

FIG. 19. Effects of insulin and Compound #43 on the glucose uptake inthe differentiated mouse C2C12 (skeletal muscle) cells. Equal number ofC2C12 cells were seeded on 96-well plates (5000 cells/well), cultured in10% FBS DMEM media for 5 days, differentiated in 0.5% horseserum-containing DMEM media for 7 days. The completely differentiatedC2C12 cells were pretreated without or with Compound #43 (300 or 600ppb) in serum/glucose-free DMEM media overnight, and then incubatedwithout (basal) or with insulin, Compound #43, or both in glucose-freeDMEM media at 37° C. for 1.5 hr. After the treatments, cells wereincubated with 1 mM of 2-deoxyglucose (2DG) at room temperature for 30minutes, and then subjected to luminescence analysis using Promega'sGlucose Uptake-Glo Assay kit. Data are presented as mean±SEM ofindicated number of samples per group. P value was derived by comparisonto the basal group.

FIG. 20. Restoration of insulin receptor function (indicated by elevatedphosphorylation of Insrβ at Tyrosine 1146) in the skeletal muscle ofinsulin-resistant Lepr^(db/db) mice after chronic treatment withCompound #43. Lepr^(db/db) mice at postnatal day 38 wereintraperitoneally injected with saline (containing 0.2% the compoundsolvent DMSO) or Compound #43 at a dose of 0.136 mg of Compound #43 perkilogram body weight daily for 52 days. Western blots were performed onskeletal muscle protein extracts (100 μg protein per lane) isolated fromsaline or Compound #43-treated Lepr^(db/db) mice. (A) Images of Westernblots. (B-C) Quantitative protein levels (normalized by β-tubulinprotein level in each sample). Data are presented as mean±SEM of fivemice. P values were calculated by comparison of treatment values tovalues in the control/saline group.

FIG. 21. Activation of Insr and stimulation of phosphorylation ofPdk1/Akt/AS160 in differentiated mouse C2C12 (skeletal muscle) cells byCompound #43. Equal number of C2C12 cells were seeded on 12-well plates(60,000 cells/well), cultured in 10% FBS DMEM media for 5 days,differentiated in 0.5% horse serum-containing DMEM media for 7 days.Completely differentiated C2C12 cells were serum-starved overnight andthen treated without or with Compound #43 (600 ppb) or insulin (200 nM)in serum-free DMEM media at 37° C. for (A-B) 5 minutes or (C-D) 30minutes, and then subjected to Western blot analysis. (A, C) Images ofWestern blots. (B, D) Quantitative protein levels (normalized byβ-tubulin protein level in each sample). Data are presented as mean±SEMof three samples per group. *P<0.05, **P<0.01, ***P<0.001 when comparedto the control group (without Compound #43 treatment).

FIG. 22. Restoration of insulin receptor function (indicated by elevatedtyrosine phosphorylation of Insrβ) in the livers of insulin-resistantLepr^(db/db) mice after chronic treatment with Compound #43.Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline (containing 0.2% the compound solvent DMSO) or Compound #43at a dose of 0.136 mg of Compound #43 per kilogram body weight daily for52 days. Liver protein extracts isolated from saline- or Compound#43-treated Lepr^(db/db) mice were subjected to ELISA assays of (A)phospho-Insrβ at Y1146 and (B) phospho-Insrβ at Y1150/1151, and Westernblot analysis of β-tubulin. (A) The protein levels of phosphor-Insrβ atY1146 (normalized by β-tubulin protein level in each sample). (B) Theprotein levels of phospho-Insrβ at Y1150/1151 (normalized by β-tubulinprotein level in each sample). Data are presented as mean±SEM of theindicated number of mice. P values were calculated by comparison oftreatment values to values in the saline group.

FIG. 23. Activation of INSR and stimulation of AS160 phosphorylation inhuman liver HepG2 cells by Compound #43. HepG2 cells were seeded on6-well plates (7×10⁵ cells/well), cultured in 10% FBS EMEM media for 30hr, and then serum-starved overnight. These serum-starved HepG2 cellswere then treated with Compound #43 (600 ppb) at 37° C. for 30 and 60minutes (min), and then subjected to Western blot analysis. (A) Imagesof Western blots. (B) Quantitative protein levels (normalized by ACTBprotein level in each sample). Data are presented as mean±SEM of threesamples per group. **P<0.01, when compared to the control group (0 mingroup, before Compound #43 treatment).

FIG. 24. Chronic treatment of Compound #43 resulted in a decrease ofserum insulin and alanine aminotransferase (ALT), but not creatinine,levels in Lepr^(db/db) mice. Male Lepr^(db/db) mice at 38 days of agewere intraperitoneally (ip) injected daily with physiological saline(0.09% NaCl) containing 0.2% DMSO, or Compound #43 at a dose of 0.136 mgper kilogram body weight, diluted in sterile physiological saline) for52 days. Sera from 3-month-old wild-type (non-diabetic) C57 mice werealso collected. These serum samples were collected and subjected to (A)insulin, (B) ALT and (C) creatinine assays. Numbers on the top of eachcolumn in (A) are the mean values of insulin levels. Different lettersin the bars represents a statistical significance between those twogroups.

FIG. 25. Mode of action of Compound #43 against type I and II diabetes.

FIG. 26. Direct activation of insulin receptor by Compound #43 andinsulin in a cell-free system. Equal amounts of native insulin receptorproteins, containing both alpha and beta subunits were incubated with0.003% DMSO (Compound #43 solvent), Compound #43 or insulin (Ins, 0.5μM) in the presence of ATP, and then subjected to Western blot analysisto detect phosphorylated Insrβ (activated Insr) at Y1146, 1150 and 1151.Different alphabetic letter in the bar graph denotes statisticallysignificant changes between those groups.

FIG. 27. Compound #68 was less effective than Compound #43 in theactivation of insulin receptor in the cell-free system. Equal amounts ofnative insulin receptor were incubated with 0.003% DMSO (Compound #43solvent), Compound #43 or Compound #68 in the presence of ATP. Theactivated Insr proteins were detected by Western blot analysis ofphosphorylated Insrβ at Y1146, 1150 and 1151. Different alphabeticletters in the bar graph mean statistically significant changes occurredbetween those groups.

FIG. 28. Reduced blood glucose levels in STZ-induced T1D mice afteracute treatment of Compound #43. STZ-induced T1D mice with unfastedblood glucose levels between 500-550 mg/dL were fasted overnight, andinjected intraperitoneally with Compound #43 at a dose of 5.4 mg/kg bodyweight or with physiological saline containing 2% DMSO (Compound #43stock solvent, referred to as Control group) for 1, 2 and 3 hours. Micewere then subjected to blood glucose measurement. P values were derivedby comparing Compound #43 treatments to the control/saline group at eachtime point.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure,chemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito: 1999, the entire contents of which are incorporatedherein by reference.

The singular forms “a”, “an”, and “the,” as used herein and in theclaims, include the plural reference unless the context clearlyindicates otherwise. Thus, for example, a reference to “a compound”includes a plurality of such compounds.

About: The term “about”, when used herein in reference to a value,refers to a value that is similar, in context to the referenced value.In general, those skilled in the art, familiar with the context, willappreciate the relevant degree of variance encompassed by “about” inthat context. For example, in some embodiments, the term “about” mayencompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless of the referred value.

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system.Those of ordinary skill in the art will be aware of a variety of routesthat may, in appropriate circumstances, be utilized for administrationto a subject, for example a human. For example, in some embodiments,administration may be ocular, oral, parenteral, topical, etc. In someparticular embodiments, administration may be bronchial (e.g., bybronchial instillation), buccal, dermal (which may be or comprise, forexample, one or more of topical to the dermis, intradermal, interdermal,transdermal, etc.), enteral, intra-arterial, intradermal, intragastric,intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal,intravenous, intraventricular, within a specific organ (e. g.intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual,topical, tracheal (e.g., by intratracheal instillation), vaginal,vitreal, etc. In some embodiments, administration may involve dosingthat is intermittent (e.g., a plurality of doses separated in time)and/or periodic (e.g., individual doses separated by a common period oftime) dosing. In some embodiments, administration may involve continuousdosing (e.g., perfusion) for at least a selected period of time.

Alzheimer's disease: As used herein, the term “Alzheimer's disease”, or“AD”, refers to a progressive disease of the human central nervoussystem. Brain insulin signaling is important for learning and memory,and insulin resistance in the brain is a major risk factor for AD. Therestoration of insulin signaling has emerged as a potential therapy forAD (White M F, Science 2003; 302:1710-1; De Felice D G et al,Alzheimer's & Dementia 2014; 10: S26-S32). In certain embodiments, it ismanifested by dementia typically in the elderly, by disorientation, lossof memory, difficulty with language, calculation, or visual-spatialskills, and by psychiatric manifestations. In certain embodiments, it isassociated with degenerating neurons in several regions of the brain.The term “dementia” as used herein includes, but is not restricted to,Alzheimer's dementia with or without psychotic symptoms. In certainembodiment, the therapeutic methods provided herein are effective forthe treatment of mild, moderate and severe Alzheimer's disease in asubject. Phases of Alzheimer's further include “moderately severecognitive decline,” also referred to as “moderate or mid-stageAlzheimer's disease,” “severe cognitive decline,” also referred to as“moderately severe or mid-stage Alzheimer's disease,” and “very severecognitive decline,” also referred to as “severe or late-stageAlzheimer's disease.” Moderately severe cognitive decline ischaracterized by major gaps in memory and deficits in cognitive functionemerge. At this stage, some assistance with day-to-day activitiesbecomes essential. In severe cognitive decline, memory difficultiescontinue to worsen, significant personality changes may emerge andaffected individuals need extensive help with customary dailyactivities. Late stage Alzheimer's disease or very severe cognitivedecline is the final stage of the disease when individuals lose theability to respond to their environment, the ability to speak and,ultimately, the ability to control movement.

Biologically activity: As used herein, the term “Biologically activity”refers to an observable biological effect or result achieved by an agentor entity of interest. For example, in some embodiments, a specificbinding interaction is a biological activity. In some embodiments,modulation (e.g., induction, enhancement, or inhibition) of a biologicalpathway or event is a biological activity. In some embodiments, presenceor extent of a biological activity is assessed through detection of adirect or indirect product produced by a biological pathway or event ofinterest.

Combination therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, the two or more regimens may beadministered simultaneously; in some embodiments, such regimens may beadministered sequentially (e.g., all “doses” of a first regimen areadministered prior to administration of any doses of a second regimen);in some embodiments, such agents are administered in overlapping dosingregimens. In some embodiments, “administration” of combination therapymay involve administration of one or more agents or modalities to asubject receiving the other agents or modalities in the combination. Forclarity, combination therapy does not require that individual agents beadministered together in a single composition (or even necessarily atthe same time), although in some embodiments, two or more agents, oractive moieties thereof, may be administered together in a combinationcomposition, or even in a combination compound (e.g., as part of asingle chemical complex or covalent entity).

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, sets of conditions, etc., that may not beidentical to one another but that are sufficiently similar to permitcomparison therebetween so that one skilled in the art will appreciatethat conclusions may reasonably be drawn based on differences orsimilarities observed. In some embodiments, comparable sets ofconditions, circumstances, individuals, or populations are characterizedby a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable. For example, those ofordinary skill in the art will appreciate that sets of circumstances,individuals, or populations are comparable to one another whencharacterized by a sufficient number and type of substantially identicalfeatures to warrant a reasonable conclusion that differences in resultsobtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicativeof the variation in those features that are varied.

Diabetes: A central characteristic of diabetes is impaired β-cellfunction. One abnormality that occurs early in disease progression inboth type I and II diabetes is the loss of eating-induced rapid insulinresponse. Consequently, the liver continues to produce glucose, whichadds to glucose that is ingested and absorbed from the basic componentsof a meal.

Type II Diabetes: One characteristic of type II diabetes is impairedinsulin action, termed insulin resistance. Insulin resistance manifestsitself as both a reduced maximal glucose elimination rate (GERmax) andan increased insulin concentration required to attain GERmax. Thus, tohandle a given glucose load more insulin is required and that increasedinsulin concentration must be maintained for a longer period of time.Consequently, the diabetic patient is also exposed to elevated glucoseconcentrations for prolonged periods of time, which further exacerbatesinsulin resistance. Additionally, prolonged elevated blood glucoselevels are themselves toxic to β-cells. Another characteristic of typeII diabetics is a delayed response to increases in blood glucose levels.While normal individuals usually begin to release insulin within 2-3minutes following consumption of food, type II diabetics may not secreteendogenous insulin until blood glucose begins to rise, and then withsecond-phase kinetics, that is a slow rise to an extended plateau inconcentration. As a result, endogenous glucose production is not shutoff and continues after consumption and the patient experienceshyperglycemia (elevated blood glucose levels). Type II diabetes arisesfrom different and less well understood circumstances. The early loss ofearly phase insulin release, and consequent continual glucose release,contributes to elevated glucose concentrations. High glucose levelspromote insulin resistance, and insulin resistance generates prolongedelevations of serum glucose concentration. This situation can lead to aself-amplifying cycle in which ever greater concentrations of insulinare less effective at controlling blood glucose levels. Moreover, asnoted above, elevated glucose levels are toxic to β-cells, reducing thenumber of functional β-cells. Genetic defects impairing the growth ormaintenance of the microvasculature nourishing the islets can also playa role in their deterioration (Glee, S. M., et al. Nature Genetics38:688-693, 2006). Eventually, the pancreas becomes overwhelmed, andindividuals progress to develop insulin deficiency similar to peoplewith type I diabetes.

Type I Diabetes: Type I diabetes occurs as a result of the destructionof insulin-producing cells of the pancreas (β-cells) by the body's ownimmune system. This ultimately results in a complete insulin hormonedeficiency.

Dosage form or unit dosage form: As used herein, the term “dosage formor unit dosage form” refers to a physically discrete unit of an activeagent (e.g., a therapeutic or diagnostic agent) for administration to asubject. In some embodiments, each such unit contains a predeterminedquantity of active agent. In some embodiments, such quantity is a unitdosage amount (or a whole fraction thereof) appropriate foradministration in accordance with a dosing regimen that has beendetermined to correlate with a desired or beneficial outcome whenadministered to a relevant population (i.e., with a therapeutic dosingregimen). Those of ordinary skill in the art appreciate that the totalamount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing regimen: As used herein, the term “dosing regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which is separatedin time from other doses. In some embodiments, individual doses areseparated from one another by a time period of the same length; in someembodiments, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. In some embodiments, different doses within a dosing regimen areof different amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount same as the first dose amount In some embodiments, a dosingregimen is correlated with a desired or beneficial outcome whenadministered across a relevant population (i.e., is a therapeutic dosingregimen).

Excipient: As used herein, the term “excipient” refers to anon-therapeutic agent that may be included in a pharmaceuticalcomposition, for example to provide or contribute to a desiredconsistency or stabilizing effect. In some embodiments, suitablepharmaceutical excipients may include, for example, starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like.

Honeymoon phase: As used herein, the term “honeymoon phase” of type 1diabetes refers to early stages of the disease characterized by loss ofearly phase insulin release and the remaining β-cell function producessome insulin, which is released with second-phase kinetics.

Hyperglycemia: As used herein, the term “Hyperglycemia” refers to adisease, disorder, or condition characterized by a higher than normalfasting blood glucose concentration. In some embodiments, hyperglycemiais characterized by a blood glucose concentration of 126 mg/dL orhigher. In some embodiments, hyperglycemia is characterized by a bloodglucose concentration of 280 mg/dL (15.6 mM) or higher.

Hypoglycemia: As used herein, the term “hypoglycemia” refers to adisease, disorder, or condition characterized by a lower than normalblood glucose concentration. In some embodiments, hypoglycemia ischaracterized by a blood glucose concentration of 63 mg/dL (3.5 mM) orlower. In some embodiments, hypoglycemia causes symptoms such ascognitive impairment, behavioral changes, pallor, diaphoresis hypotonia,flush and weakness that are recognized symptoms of hypoglycemia and thatdisappear with appropriate caloric intake. In some embodiments,hypoglycemia is severe such that glucagon injections, glucose infusions,or help by another party are required.

Improve, increase, inhibit or reduce: As used herein, the terms“improve”, “increase”, “inhibit’, and “reduce”, or grammaticalequivalents thereof, indicate values that are relative to a baseline orother reference measurement. In some embodiments, an appropriatereference measurement may be or comprise a measurement in a particularsystem (e.g., in a single individual) under otherwise comparableconditions absent presence of (e.g., prior to and/or after) a particularagent or treatment, or in presence of an appropriate comparablereference agent. In some embodiments, an appropriate referencemeasurement may be or comprise a measurement in comparable system knownor expected to respond in a particular way, in presence of the relevantagent or treatment.

Insulin-related disorder: As used herein, the term “insulin-relateddisorders” refers to disorders involving production, regulation,metabolism, and action of insulin in a mammal. Insulin-related disordersinclude, but are not limited to, pre-diabetes, type I diabetes, type IIdiabetes, hypoglycemia, hyperglycemia, insulin resistance, secretorydysfunction, sarcopenia, loss of pancreatic β-cell function, and loss ofpancreatic β-cells.

Non-insulin dependent patients having insulin-related disorders: As usedherein, the term “non-insulin dependent patients having insulin-relateddisorders” refers to patients with disorders for which therapy withexogenously-provided insulin is not the current standard treatment upondiagnosis. Non-insulin dependent patients having insulin-relateddisorders which are not treated with exogenously-administered insulininclude early type II diabetes, type I diabetes in the honeymoon phase,pre-diabetes and insulin-producing cell transplant recipients.

Insulin resistance: As used herein, the term “insulin resistance” refersto the inability of a patient's cells to respond to insulinappropriately or efficiently. The pancreas responds to this problem atthe cellular level by producing more insulin. Eventually, the pancreascannot keep up with the body's need for insulin and excess glucosebuilds up in the bloodstream. Patients with insulin resistance oftenhave high levels of blood glucose and high levels of insulin circulatingin their blood at the same time.

Insulin resistance disorder: As used herein, the term “insulinresistance disorder” refers to any disease or condition that is causedby or contributed to by insulin resistance. Examples include: diabetes,obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X,insulin resistance, high blood pressure, hypertension, high bloodcholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atheroscleroticdisease including stroke, coronary artery disease or myocardialinfarction, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia,impaired glucose tolerance, delayed insulin release, diabeticcomplications, including coronary heart disease, angina pectoris,congestive heart failure, stroke, cognitive functions in dementia,retinopathy, neuropathy, nephropathy, glomerulonephritis,glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosissome types of cancer (such as endometrial, breast, prostate, and colon),complications of pregnancy, poor female reproductive health (such asmenstrual irregularities, infertility, irregular ovulation, polycysticovarian syndrome (PCOS)), lipodystrophy, cholesterol related disorders,such as gallstones, cholecystitis and cholelithiasis, gout, obstructivesleep apnea and respiratory problems, osteoarthritis, and prevention andtreatment of bone loss, e.g. osteoporosis.

Isomer: As is known in the art, many chemical entities (in particularmany organic molecules and/or many small molecules) can exist in avariety of structural (e.g., geometric, conformational, isotopic) and/oroptical isomeric forms. For example, any chiral center can exist in Rand S configurations, double bonds can exist in Z and E conformationalisomers, certain structural elements can adopt two or more tautomericforms, certain structures can be substituted with one or moreisotopically enriched atoms (e.g., deuterium or tritium for hydrogen,¹²C or ¹⁴C for ¹³C, ¹³¹I for ¹²⁹I, etc.). In some embodiments, as willbe clear to those skilled in the art from context, depiction of orreference to a particular compound structure herein may represent allstructural and/or optical isomers thereof. In some embodiments, as willbe clear to those skilled in the art from context, depiction of orreference to a particular compound structure herein is intended toencompass only the depicted or referenced isomeric form. In someembodiments, compositions including a chemical entity that can exist ina variety of isomeric forms include a plurality of such forms; in someembodiments such compositions include only a single form. For example,in some embodiments, compositions including a chemical entity that canexist as a variety of optical isomers (e.g., stereoisomers,diastereomers, etc.) include a racemic population of such opticalisomers; in some embodiments such compositions include only a singleoptical isomer and/or include a plurality of optical isomers thattogether retain optical activity.

Parenteral: As used herein, the terms “parenteral administration” and“administered parenterally” refer to modes of administration other thanenteral and topical administration, usually by injection. In someembodiments, parenteral administration may be or comprise intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and/or infusion.

Partially Unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, the active agent is present in unit doseamount appropriate for administration in a therapeutic regimen thatshows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. In some embodiments, a pharmaceutical composition may bespecially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces. Those skilled in the art willappreciate that, in general, any composition that is formulated foradministration to a human or animal subject, may, in some embodiments,be considered to be a pharmaceutical composition, whether or not itsadministration requires a medical prescription. Thus, for example, insome embodiments, a food or food supplement composition (e.g., a liquidor solid consumable composition such as a shake or sports drink ornutritional supplement powder) may be considered to be a pharmaceuticalcomposition. Alternatively or additionally, in some embodiments, apharmaceutical composition may be a formulation that is specificallyregulated and approved for administration to relevant subjects by anappropriate government agency such as, for example, the Food and DrugAdministration in the United States. In some embodiments, apharmaceutical composition is one that cannot legally be administeredwithout a prescription from a licensed medical practitioner.

Pharmaceutically acceptable: As used herein, the term “pharmaceuticallyacceptable” applied to a carrier, diluent, or excipient used toformulate a composition as disclosed herein means that the carrier,diluent, or excipient must be compatible with the other ingredients ofthe composition and not deleterious to the recipient thereof.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” refers to apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pre-Diabetes: As used herein, the term “pre-diabetes” refers to adisease, disorder, or condition where the patients have impaired fastingglucose, and/or impaired glucose tolerance. In some embodiments,pre-diabetic patients have a fasting blood glucose level between 100mg/dL (5.5 mmol/L) and 126 mg/dL (7.0 mmol/L). In some embodiments,pre-diabetic patients have a 2 hour post-prandial blood glucose levelbetween 140 mg/dL (7.8 mmol/L) and 200 mg/dL (11.1 mmol/L).

Prodrug: As used herein, the term “prodrug” refers to apharmacologically active or more typically an inactive compound that isconverted into a pharmacologically active agent by a metabolictransformation. Prodrugs of a compound of any of the formulas asdescribed herein are prepared by modifying functional groups present inthe compound of any of the formulas in such a way that the modificationsmay be cleaved in vivo to release the parent compound. In vivo, aprodrug readily undergoes chemical changes under physiologicalconditions (e.g., are hydrolyzed or acted on by naturally occurringenzyme(s)) resulting in liberation of the pharmacologically activeagent. Prodrugs include compounds of any of the formulas as describedherein wherein a hydroxyl, amino, or carboxyl group is bonded to anygroup that may be cleaved in vivo to regenerate the free hydroxyl, aminoor carboxyl group, respectively. Examples of prodrugs include, but arenot limited to esters (e.g., acetate, formate, and benzoate derivatives)of compounds of any of the formulas as described herein or any otherderivative which upon being brought to the physiological pH or throughenzyme action is converted to the active parent drug. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described in the art (see, for example, Bundgaard.Design of Prodrugs. Elsevier, 1985).

Proliferative condition: As used herein, the term “proliferativecondition” refers to a disease or disorder associated with cellproliferation. In some embodiments, a proliferative disease or disorderis or comprises cancer. In some embodiments, a proliferative disease ordisorder is an inflammatory disease or disorder. In some embodiments, aproliferative disease or disorder is an autoimmune disease or disorder.In some embodiments, a proliferative disease or disorder is a microbialinfection (e.g., a bacterial infection).

Reference: As used herein, the term “reference” refers to a standard orcontrol relative to which a comparison is performed. For example, insome embodiments, an agent, animal, individual, population, sample,sequence or value of interest is compared with a reference or controlagent, animal, individual, population, sample, sequence or value. Insome embodiments, a reference or control is tested and/or determinedsubstantially simultaneously with the testing or determination ofinterest. In some embodiments, a reference or control is a historicalreference or control, optionally embodied in a tangible medium.Typically, as would be understood by those skilled in the art, areference or control is determined or characterized under comparableconditions or circumstances to those under assessment. Those skilled inthe art will appreciate when sufficient similarities are present tojustify reliance on and/or comparison to a particular possible referenceor control.

Risk: As used herein, the “risk” of a disease, disorder, and/orcondition refers to the likelihood that a particular individual willdevelop a disease, disorder, and/or condition. In some embodiments, riskis expressed as a percentage. In some embodiments, risk is from 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. Insome embodiments risk is expressed as a risk relative to a riskassociated with a reference sample or group of reference samples. Insome embodiments, a reference sample or group of reference samples havea known risk of a disease, disorder, condition and/or event. In someembodiments a reference sample or group of reference samples are fromindividuals comparable to a particular individual. In some embodiments,relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Solid form: As is known in the art, many chemical entities (inparticular many organic molecules and/or many small molecules) can adopta variety of different solid forms such as, for example, amorphous formsand/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.).In some embodiments, such entities may be utilized as a single such form(e.g., as a pure preparation of a single polymorph). In someembodiments, such entities may be utilized as a mixture of such forms.

Subject: As used herein, the term “subject” refers an organism,typically a mammal (e.g., a human, in some embodiments includingprenatal human forms). In some embodiments, a subject is suffering froma relevant disease, disorder or condition. In some embodiments, asubject is susceptible to a disease, disorder, or condition. In someembodiments, a subject displays one or more symptoms or characteristicsof a disease, disorder or condition. In some embodiments, a subject doesnot display any symptom or characteristic of a disease, disorder, orcondition. In some embodiments, a subject is someone with one or morefeatures characteristic of susceptibility to or risk of a disease,disorder, or condition. In some embodiments, a subject is a patient. Insome embodiments, a subject is an individual to whom diagnosis and/ortherapy is and/or has been administered.

Susceptible to: An individual who is “susceptible to” a disease,disorder, or condition (e.g., influenza) is at risk for developing thedisease, disorder, or condition. In some embodiments, an individual whois susceptible to a disease, disorder, or condition does not display anysymptoms of the disease, disorder, or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, or condition hasnot been diagnosed with the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder, orcondition is an individual who has been exposed to conditions associatedwith development of the disease, disorder, or condition. In someembodiments, a risk of developing a disease, disorder, and/or conditionis a population-based risk (e.g., family members of individualssuffering from the disease, disorder, or condition).

Therapeutically effective amount: As used herein, the term“Therapeutically effective amount” refers to an amount that produces thedesired effect for which it is administered. In some embodiments, theterm refers to an amount that is sufficient, when administered to apopulation suffering from or susceptible to a disease, disorder, and/orcondition in accordance with a therapeutic dosing regimen, to treat thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is one that reduces the incidenceand/or severity of, and/or delays onset of, one or more symptoms of thedisease, disorder, and/or condition. Those of ordinary skill in the artwill appreciate that the term “therapeutically effective amount” doesnot in fact require successful treatment be achieved in a particularindividual. Rather, a therapeutically effective amount may be thatamount that provides a particular desired pharmacological response in asignificant number of subjects when administered to patients in need ofsuch treatment. In some embodiments, reference to a therapeuticallyeffective amount may be a reference to an amount as measured in one ormore specific tissues (e.g., a tissue affected by the disease, disorderor condition) or fluids (e.g., blood, saliva, serum, sweat, tears,urine, etc.). Those of ordinary skill in the art will appreciate that,in some embodiments, a therapeutically effective amount of a particularagent or therapy may be formulated and/or administered in a single dose.In some embodiments, a therapeutically effective agent may be formulatedand/or administered in a plurality of doses, for example, as part of adosing regimen.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to administration of a therapy that partially orcompletely alleviates, ameliorates, relieves, inhibits, delays onset of(e.g., relative to an established onset time or period), reducesseverity of, and/or reduces incidence of one or more symptoms, features,and/or causes of a particular disease, disorder, and/or condition. Insome embodiments, treatment may be of a subject who does not exhibitsigns or symptoms of the relevant disease, disorder and/or condition,and/or of a subject who is not diagnosed suffering the relevant disease,disorder and/or condition, and/or of a subject who exhibits only earlysigns of the disease, disorder, and/or condition. Alternatively oradditionally, in some embodiments, treatment may be of a subject whoexhibits one or more established signs of the relevant disease, disorderand/or condition. In some embodiments, treatment may be of a subject whohas been diagnosed as suffering from the relevant disease, disorder,and/or condition. In some embodiments, treatment may be of a subjectknown to have one or more susceptibility factors that are statisticallycorrelated with increased risk of development of the relevant disease,disorder, and/or condition. In some embodiments, such treatment refersto reducing risk of developing the disease, disorder and/or conditionand/or to delaying onset of one or more characteristics or symptoms ofthe disease, disorder or condition. In some embodiments, treatment isadministration of therapy according to a regimen that has beendemonstrated to achieve a relevant result (e.g., to partially orcompletely alleviate, ameliorate, relieve, inhibit, delay onset of,reduce severity of, and/or reduce incidence of one or more symptoms,features, and/or cause of a particular disease, disorder, and/orcondition) with statistical significance when applied to a relevantpopulation or system (e.g., model system). In some embodiments,treatment administered after diagnosis and/or onset of one or moresymptoms is considered to be “therapeutic” treatment, whereas treatmentadministered prior to diagnosis and/or to onset of symptoms isconsidered to be “prophylactic” treatment.

Unit dose: As used herein, the term “unit dose” refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the, attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Alkyl: As used herein, the term “alkyl” refers to linear or branchedalkyl groups. Exemplary C₁₋₆alkyl groups include, but are not limitedto, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,pentyl, isopentyl, neopentyl, and hexyl.

Heteroatom: As used herein, the term “heteroatom” refers to one or moreof oxygen, sulfur, nitrogen, phosphorus, or silicon (including, anyoxidized form of nitrogen, sulfur, phosphorus, or silicon; thequaternized form of any basic nitrogen or; a substitutable nitrogen of aheterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

Carbocyclic: As used herein, the term “carbocyclic” refers to amonocyclic hydrocarbon that is completely saturated or that contains oneor more units of unsaturation, but which is not aromatic, that has asingle point of attachment to the rest of the molecule.

Heterocyclic: As used herein, the term “heterocyclic” refers to a stablemonocyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl). Aheterocyclic ring can be attached to its pendant group at any heteroatomor carbon atom that results in a stable structure and any of the ringatoms can be optionally substituted. Examples of 3-8 memberedheterocyclic include tetrahydrofuranyl, tetrahydrothiophenyl,pyrrolidinyl, piperidinyl, pyrrolinyl, oxazolidinyl, piperazinyl,dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, andmorpholinyl.

Halogen: As used herein, the terms “halogen” and “halo” refer to F, Cl,Br, or I.

Compound C: The term “Compound C”, as used herein, refers to5′-Methylselenoadenosine; also known as (2R, 4S,5S)-2-(6-amino-9H-purin-9-yl)-5-((methylselanyl)methyl)tetrahydrofuran-3,4-diol,CAS Registry Number 5135-40-0, and includes any pharmaceuticallyacceptable salts thereof.

Compound D: The term “Compound D”, as used herein, refers to5′-Selenoadenosyl homocysteine; also known as(2R)-2-amino-4-((((2S,3S,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)selanyl)butanoicacid, CAS Registry Number 4053-91-2, and includes any pharmaceuticallyacceptable salts thereof.

Compound E: The term “Compound E”, as used herein, refers togamma-glutamyl-methylseleno-cysteine orγ-L-glutamyl-Se-methyl-L-cysteine; also known asN5-(1-carboxy-2-(methylselanyl)ethyl)-L-glutamine, or anypharmaceutically acceptable salt thereof.

Compound CDE: The term “Compound CDE”, as used herein, refers to amixture of Compound C, Compound D, and Compound E, or pharmaceuticallyacceptable salts thereof.

Compounds

The present disclosure provides a number of exemplary compounds capableof lowering glucose level, improving glucose tolerance, restoring oractivating insulin receptor function and its downstream signaling,enhancing AS160 phosphorylation for translocation of glucose transporterproteins (GLUTs) from cytosolic vesicles to plasma membrane for glucoseuptake, stimulating glucose uptake, and attenuating hyperinsulinemiawithout impaired kidney function and/or liver damage in diabetic mice,cultured liver and/or skeletal muscle cells after treatment of singlecompound. For example, Example 2 shows that each of Compounds 43, 50,53, 69, and 70 lowered glucose production in HepG2 cells. Particularly,Compound 43 exhibited higher potency than Compound CDE. When compared toantidiabetic drug metformin in HepG2 and rat H4IIE cells, Compound 43showed greater potency and lower cell toxicity. It was also shown thatCompound #43 is more potent than Compound 50 in the inhibition of theexpression of G6pc (a key enzyme gene for liver glucose production) inthe liver of diabetic mice, and than Compound CDE, Compound C, CompoundD and Compound 50 in mouse liver AML-12 cells (Example 4). Further, thepresent disclosure demonstrates that compound 43 is more potent thancompound C, compound 50, compound 69 and compound 70 againsthyperglycemia in diabetic mice (Example 3); that Compound 43significantly improves glucose tolerance (Example 3) andenhances/restores insulin receptor function and its downstream signalingin the livers and skeletal muscles of insulin-resistant diabeticLepr^(db/db) mice (Examples 5 and 7-8); and that Compound 43 elicits aresponse to the glucose challenge in diabetic mice which was similar tothe response in wild-type mice (Example 3). In addition, the presentdisclosure demonstrates that Compound #43 treatment can enhance thephosphorylation of AS160 (to promote the translocation of GLUTs fromcellular vesicles to plasma membrane) for glucose uptake in both liverand skeletal muscle cells (Example 8), stimulate GLUT4 expression in theliver of diabetic mice and cultured mouse liver cells (Example 6),enhance and/or potentiate insulin action to stimulate glucose uptake inthe liver and skeletal muscle cells (Example 6-7), and attenuate thehyperinsulinemia without impaired kidney function and/or liver damage inthe insulin-resistant diabetic mice (Example 9).

The present disclosure also provides features of selenium compoundswhich may contribute to its activity. For example, it was shown thatwhile selenium Compound #43 had a great potency in inhibiting glucoseproduction, its sulfur analog Compound 68 had a low potency (Example 2);and that Compound #43 is more potent than Compound #68 in lowering bloodglucose and HbA1c levels and improving glucose tolerance ininsulin-resistant diabetic mice (Example 3). It was also shown thatinhibition of glucose production was observed after treatment ofselenium compounds comprising, at 2′ and 3′ position, diacetyl ester(#43), cyclic carbonate (Compound #50), morpholino carboxylate (#53),dipropanoyl ester (#69), or dibutanoyl ester (#70), with the greatestpotency observed after treatment of Compound 43 (Example 2). Further, itwas shown that compounds comprising a 5′ methyl seleno group and a 5′seleno homocysteine group had similar potency in inhibiting glucoseproduction in HepG2 cells (compound C vs. compound D in Example 2). Invivo studies also revealed that Compound #43 is more potent thanCompound #69 and #70 in lowering blood glucose levels and improvingglucose tolerance in insulin-resistant diabetic mice (Example 3).

In some embodiments, the present disclosure relates to a compound offormula (1):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,wherein

each of R² and R³ is independently H or —C(O)—R, wherein each R isindependently C₁₋₆alkyl or 3-8 membered carbocyclic or heterocyclic,wherein R² and R³ cannot be both H;

or R² together with R³ form —(CH₂)_(n)—C(O)—(CH₂)_(m)—, wherein each ofn and m is independently 0-3, and n+m≤3;

R⁵ is —C₁₋₆alkyl or —C₁₋₆alkyl-CH(NH₂)COOH;

R⁸ is H or halogen; and

X is H or halogen;

wherein each of the carbocyclic, heterocyclic, —(CH₂)_(n)—, and—(CH₂)_(m)— moieties, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and

each C₁₋₆alkyl moiety, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, or CN.

In some embodiments of formula (1), R⁸ is H. In some embodiments offormula (1), R⁸ of formula (1) is halogen. In some embodiments offormula (1), R⁸ of formula (1) is F.

In some embodiments of formula (1), X is H. In some embodiments offormula (1), X is halogen. In some embodiments of formula (1), X is F.

In some embodiments of formula (1), R⁵ is —C₁₋₆alkyl, which mayoptionally be substituted 1-3 times by —OH, halogen, NH₂, or CN. In someembodiments of formula (1), R⁵ is —C₁₋₆alkyl, which may optionally besubstituted 1-3 times by halogen. In some embodiments of formula (1), R⁵is unsubstituted —C₁₋₆alkyl. In some embodiments of formula (1), R⁵ isunsubstituted linear —C₁₋₆alkyl. In some embodiments of formula (1), R⁵is methyl. In some embodiments of formula (1), R⁵ is ethyl. In someembodiments of formula (1), R⁵ is propyl.

In some embodiments of formula (1), R⁵ is —C₁₋₆alkyl-CH(NH₂)COOH,wherein C₁₋₆alkyl may optionally be substituted 1-3 times by —OH,halogen, NH₂, or CN. In some embodiments of formula (1), R⁵ is—C₁₋₆alkyl-CH(NH₂)COOH, wherein C₁₋₆alkyl may optionally be substituted1-3 times by halogen. In some embodiments of formula (1), R⁵ is—C₁₋₆alkyl-CH(NH₂)COOH, wherein C₁₋₆alkyl is unsubstituted. In someembodiments of formula (1), R⁵ is —CH₂CH₂—CH(NH₂)COOH. In someembodiments of formula (1), R⁵ is —CH₂—CH(NH₂)COOH. In some embodimentsof formula (1), R⁵ is —CH₂CH₂CH₂—CH(NH₂)COOH.

In some embodiments of formula (1), R² is H, and R³ is —C(O)—R, whereinR is C₁₋₆ alkyl or 3-8 membered carbocyclic or heterocyclic, whereineach of the carbocyclic and heterocyclic moieties, independently, mayoptionally be substituted 1-3 times by —OH, halogen, NH₂, CN, orC₁₋₆alkyl; and each C₁₋₆alkyl, independently, may optionally besubstituted 1-3 times by —OH, halogen, NH₂, or CN.

In some embodiments of formula (1), R³ is H, and R² of formula (1) is—C(O)—R, wherein R is C₁₋₆alkyl or 3-8 membered carbocyclic orheterocyclic, wherein each of the carbocyclic and heterocyclic moieties,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl, independently, may optionallybe substituted 1-3 times by —OH, halogen, NH₂, or CN.

In some embodiments of formula (1), each of R² and R³ is independentlyC(O)—R, wherein each R is independently C₁₋₆alkyl or 3-8 memberedcarbocyclic or heterocyclic, wherein each of the carbocyclic andheterocyclic moieties, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, or CN.

In some embodiments of formula (1), each R is independently 3-8 memberedcarbocyclic or heterocyclic, wherein each of the carbocyclic andheterocyclic moieties, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, or CN. In some embodiments of formula (1), each R is independently3-8 membered carbocyclic or heterocyclic, wherein each of thecarbocyclic and heterocyclic moieties, independently, may optionally besubstituted 1-3 times by halogen. In some embodiments of formula (1),each R is independently 3-8 membered carbocyclic or heterocyclic,wherein each of the carbocyclic and heterocyclic moieties,independently, may optionally be substituted 1-3 times by halogen. Insome embodiments of formula (1), each R is independently 3-8 memberedunsubstituted carbocyclic or unsubstituted heterocyclic. In someembodiments of formula (1), each R is independently 6 memberedunsubstituted carbocyclic or unsubstituted heterocyclic. In someembodiments of formula (1), each R is independently unsubstitutedheterocyclic. In some embodiments of formula (1), R is

In some embodiments of formula (1), each R is independently C₁₋₆alkyl,and each C₁₋₆alkyl, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, or CN. In some embodiments of formula (1),each R is independently C₁₋₆alkyl, and each C₁₋₆alkyl, independently,may optionally be substituted 1-3 times by halogen. In some embodimentsof formula (1), each R is independently unsubstituted C₁₋₆alkyl. In someembodiments of formula (1), each R is independently unsubstituted linearC₁₋₆alkyl. In some embodiments of formula (1), each R is independentlymethyl, ethyl, or propyl. In some embodiments of formula (1), R is CH₃.

In some embodiments of formula (1), R² together with R³ form—(CH₂)_(n)—C(O)—(CH₂)_(m)—, wherein each of n and m is independently0-3, and n+m≤3, wherein each of the —(CH₂)_(n)— and —(CH₂)_(m)—moieties, independently, may optionally be substituted 1-3 times by —OH,halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl, independently, mayoptionally be substituted 1-3 times by —OH, halogen, NH₂, or CN. In someembodiments of formula (1), each of the —(CH₂)_(n)— and —(CH₂)_(m)—moieties, independently, may optionally be substituted 1-3 times byhalogen. In some embodiments of formula (1), the —(CH₂)_(n)— and—(CH₂)_(m)— moieties are unsubstituted. In some embodiments, n=m=0.

In some embodiments, the present disclosure relates to a compound offormula (2):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,

wherein R⁸ is H or halogen;

X is H or halogen;

each R₅′ is independently H or halogen; and

each R is independently C₁₋₆alkyl, each of which, independently, mayoptionally be substituted 1-3 times by halogen.

In some embodiments of formula (2), C(R₅′)₃ is CF₃, CHF₂, CH₂F, or CH₃.

In some embodiments of formula (2), the compound is of formula (2′):

In some embodiments of formula (2) or (2′), R⁸ is H. In some embodimentsof formula (2) or (2′), R⁸ is halogen. In some embodiments of formula(2) or (2′), R⁸ is F.

In some embodiments of formula (2) or (2′), X is H. In some embodimentsof formula (2) or (2′), X is halogen. In some embodiments of formula (2)or (2′), X is F.

In some embodiments of formula (2) or (2′), R is each independentlyunsubstituted C₁₋₆alkyl. In some embodiments of formula (2) or (2′), Ris each independently C₁₋₃alkyl, each of which, independently, mayoptionally be substituted 1-3 times by halogen. In some embodiments offormula (2) or (2′), R is each independently unsubstituted C₁₋₃alkyl. Insome embodiments of formula (2) or (2′), R is each independently —CH₃,—CH₂CH₃, or —CH₂CH₂CH₃.

In some embodiments, the present disclosure relates to a compound offormula (3):

or a pharmaceutically acceptable salt, prodrug, or isomer thereof,

wherein R⁸ is H or halogen;

X is H or halogen; and

each R′ is independently H or halogen.

In some embodiments of formula (3), —Se—C(R′)₃ is —Se—CH₃, —Se—CHF₂,—Se—CH₂F, or —Se—CF₃.

In some embodiments of formula (3), the compound is of formula (3′):

In some embodiments of formula (3) or (3′), R⁸ is H. In some embodimentsof formula (3) or (3′), R⁸ is halogen. In some embodiments of formula(3) or (3′), R⁸ is F.

In some embodiments of formula (3) or (3′), X is H. In some embodimentsof formula (3) or (3′), X is halogen. In some embodiments of formula (3)or (3′), X is F.

In some embodiments of formula (3) or (3′), each C(R′)₃ is independentlyCF₃, CHF₂, or CH₂F, or CH₃. In some embodiments of formula (3) or (3′),each C(R′)₃ is independently CH₂F or CH₃. In some embodiments of formula(3) or (3′), each C(R′)₃ is CH₃.

In some embodiments, the present disclosure relates to a compound offormula:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof.Formulations

In some embodiments, the present disclosure provides compositions thatcomprise and/or deliver (i.e., upon administration to a system orsubject) a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, the present disclosure provides compositions comprisingonly a single compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, the present disclosure provides compositions comprising oneor more compounds of any one of formulas (1)-(3), or a pharmaceuticallyacceptable salt, prodrug, or isomer thereof, and one or more carriers orexcipients appropriate for administration to human or animal subjects inaccordance with the present disclosure.

In some embodiments, the present disclosure provides compositions thatdeliver an active moiety of a compound of any one of formulas (1)-(3).In some embodiments, the composition comprises an active metabolite of acompound of any one of formulas (1)-(3). In some embodiments, thecomposition comprises a compound which forms a metabolite of a compoundof any one of formulas (1)-(3) upon administration of said composition,which metabolite maintains relevant biological activity.

In some embodiments, the present disclosure provides compositions thatare pharmaceutical compositions in that they contain an activepharmaceutical ingredient (API) and one or more pharmaceuticallyacceptable carriers or excipients. In some embodiments, the API is orcomprises the compound of any one of formulas (1)-(3). In someembodiments, the API consists of the compound of any one of formulas(1)-(3). In some embodiments, the API consists of a single compound ofany one of formulas (1)-(3).

In some embodiments, the present disclosure provides methods ofmanufacturing a provided composition, for example by combining one ormore appropriate (i.e., pharmaceutically acceptable) carriers orexcipients with a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, the one or more pharmaceutically acceptable carriers orexcipients are suitable for oral administration and the mixture isformulated into an oral formulation. In some embodiments, thepharmaceutical composition is a solid dosage form. In some embodiments,the solid dosage form is a tablet, capsule, or lozenge. In someembodiments, the pharmaceutical composition is a liquid dosage form(e.g., a drink).

Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980) discloses various carriers usedin formulating pharmaceutical compositions and known techniques for thepreparation thereof. In some embodiments, the present disclosureprovides pharmaceutical compositions comprising a pharmaceuticallyacceptable amount of a compound as described herein. In someembodiments, amount of active ingredient which can be combined with acarrier material to produce a single dosage form may vary depending uponthe host being treated, and/or the particular mode of administration.The amount of active ingredient that can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally, thisamount will range from about 1% to about 99% of active ingredient, fromabout 5% to about 70%, or from about 10% to about 30%.

In some embodiments, wetting agents, emulsifiers, and lubricants, suchas sodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

In some embodiments, formulations of the present invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. In some embodiments,the formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Insome embodiments, formulations as described herein comprise an excipientselected from the group consisting of cyclodextrins, liposomes, micelleforming agents, e.g., bile acids, and polymeric carriers, e.g.,polyesters and polyanhydrides; and a compound as described herein. Insome embodiments, formulations as described herein render orallybioavailable a compound as described herein.

In some embodiments, methods of preparing such formulations may comprisea step of bringing into association a compound as described herein withone or more pharmaceutically acceptable carriers or excipients, andoptionally one or more accessory ingredients. In some embodiments, theformulations are prepared by uniformly and intimately bringing intoassociation a compound as described herein with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

In some embodiments, formulations as described herein suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes, drinks, and the like, each containing a predeterminedamount of a compound as described herein as an active ingredient. Insome embodiments, a compound as described herein may alternatively oradditionally be administered as a bolus, electuary or paste.

In some embodiments, in solid dosage forms as described herein for oraladministration (capsules, tablets, pills, dragees, powders, granules andthe like), the active ingredient is mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: fillers or extenders,such as starches, lactose, sucrose, glucose, mannitol, and/or silicicacid; binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as, for example, cetyl alcohol, glycerol monostearate, andnon-ionic surfactants; absorbents, such as kaolin and bentonite clay;lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; andcoloring agents. In some embodiments, in the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. In some embodiments, solid compositions of a similar type mayalso be employed as fillers in soft and hard-shelled gelatin capsulesusing such carriers as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

In some embodiments, a tablet may be made by compression or molding,optionally with one or more accessory ingredients. In some embodiments,compressed tablets may be prepared using binder (for example, gelatin orhydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (for example, sodium starch glycolate or cross-linkedsodium carboxymethyl cellulose), surface-active or dispersing agent. Insome embodiments, molded tablets may be made in a suitable machine inwhich a mixture of the powdered compound is moistened with an inertliquid diluent.

In some embodiments, the tablets, and other solid dosage forms of thepharmaceutical compositions as described herein, such as dragees,capsules, pills and granules, may optionally be scored or prepared withcoatings and shells, such as enteric coatings and other coatings wellknown in the pharmaceutical-formulating art. In some embodiments, theymay be formulated so as to provide slow or controlled release of theactive ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile,other polymer matrices, liposomes and/or microspheres. In someembodiments, they may be formulated for rapid release, e.g.,freeze-dried. In some embodiments, they may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. In some embodiments,these compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes. In some embodiments,the active ingredient can be in micro-encapsulated form, if appropriate,with one or more of the above-described excipients.

In some embodiments, liquid dosage forms for oral administration of thecompounds as described herein include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In some embodiments, in addition to the active ingredient, the liquiddosage forms may contain inert diluents commonly used in the art, suchas, for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

In some embodiments, besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming and preservativeagents.

In some embodiments, suspensions, in addition to the active compounds,may contain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

In some embodiments, formulations as described herein for rectal orvaginal administration may be presented as a suppository, which may beprepared by mixing one or more compounds as described herein with one ormore suitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the rectum or vaginal cavityand release the active compound.

In some embodiments, formulations as described herein which are suitablefor vaginal administration include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

In some embodiments, dosage forms for the topical or transdermaladministration of a compound as described herein include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. In some embodiments, the active compound may be mixed understerile conditions with a pharmaceutically-acceptable carrier, and withany preservatives, buffers, or propellants which may be required.

In some embodiments, the ointments, pastes, creams and gels may contain,in addition to a compound as described herein, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

In some embodiments, powders and sprays can contain, in addition to acompound as described herein, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. In some embodiments, sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

In some embodiments, transdermal patches have the added advantage ofproviding controlled delivery of a compound as described herein to thebody. In some embodiments, dissolving or dispersing the compound in theproper medium can make such dosage forms. In some embodiments,absorption enhancers can be used to increase the flux of the compoundacross the skin. In some embodiments, either providing a ratecontrolling membrane or dispersing the compound in a polymer matrix orgel can control the rate of such flux.

In some embodiments, the present disclosure provides ophthalmicformulations, eye ointments, powders, solutions and the like.

In some embodiments, pharmaceutical compositions as described hereinsuitable for parenteral administration comprise one or more compounds asdescribed herein in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use, which may contain sugars, alcohols, antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions as described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. In some embodiments, proper fluidity can be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In some embodiments, the compositions as described herein may containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. In some embodiments, prevention of the action ofmicroorganisms upon the subject compounds may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. In someembodiments, it may be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. In someembodiments, prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorptionsuch as aluminum monostearate and gelatin.

In some embodiments, for example in order to prolong the effect of adrug, it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection. In some embodiments, this maybe accomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility. In some embodiments,the rate of absorption of the drug then depends upon its rate ofdissolution, which in turn, may depend upon crystal size and crystallineform. In some embodiments, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

In some embodiments, injectable depot forms are made by formingmicroencapsule matrices of the subject compounds in biodegradablepolymers such as polylactide-polyglycolide. In some embodiments,depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). In some embodiments, depot injectable formulations areprepared by entrapping the drug in liposomes or microemulsions, whichare compatible with body tissue.

In some embodiments, drug-eluting forms include coated or medicatedstents and implantable devices. In some embodiments, drug-eluting stentsand other devices may be coated with a compound or pharmaceuticalpreparation and may further comprise a polymer designed fortime-release.

In some embodiments, a compound or pharmaceutical preparation isadministered orally. In some embodiments, the compound or pharmaceuticalpreparation is administered intravenously. In some embodiments, acompound is attached via a cleavable linker to a solid support that isadministered with a catheter. In some embodiments, routes ofadministration include sublingual, intramuscular, and transdermaladministrations.

In some embodiments, the compounds as described herein are administeredas pharmaceuticals, to humans and animals, they can be given per se oras a pharmaceutical composition containing, for example, 0.1% to 99.5%,or 0.5% to 90%, of active ingredient in combination with apharmaceutically acceptable carrier.

In some embodiments, the compounds as described herein may be givenorally, parenterally, topically, or rectally. In some embodiments, theyare of course given in forms suitable for each administration route. Insome embodiments, they are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, suppository, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories.

In some embodiments, the compounds as described herein may beadministered to humans and other animals for therapy by any suitableroute of administration, including orally, nasally, as by, for example,an aerosol, a spray, rectally, intravaginally, parenterally,intracisternally and topically, as by powders, ointments or drops,including buccally and sublingually.

In some embodiments, the compounds as described herein, which may beused in a suitable hydrated form, and/or the pharmaceutical compositionsas described herein, are formulated into pharmaceutically-acceptabledosage forms by conventional methods known to those of skill in the art.

In some embodiments, actual dosage levels of the active ingredients inthe pharmaceutical compositions as described herein may be varied so asto obtain an amount of the active ingredient that is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient.

In some embodiments, a selected dosage level will depend upon a varietyof factors including the activity of the particular compound asdescribed herein, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art candetermine and prescribe the effective amount of the pharmaceuticalcomposition required. In some embodiments, the physician or veterinariancould start doses of the compounds as described herein in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, compounds or pharmaceutical compositions asdescribed herein are provided to a subject chronically. In someembodiments, chronic treatments include any form of repeatedadministration for an extended period of time, such as repeatedadministrations for one or more months, between a month and a year, oneor more years, or longer. In some embodiments, a chronic treatmentinvolves administering a compound or pharmaceutical composition asdescribed herein repeatedly over the life of the subject. In someembodiments, chronic treatments involve regular administrations, forexample one or more times a day, one or more times a week, or one ormore times a month. In some embodiments, a suitable dose such as a dailydose of a compound as described herein will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed herein. In some embodiments, doses of the compounds asdescribed herein for a patient, when used for the indicated effects,will range from about 0.0001 to about 100 mg per kg of body weight perday. In some embodiments, the daily dosage will range from 0.001 to 50mg of compound per kg of body weight. In some embodiments, the dailydosage will range from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

In some embodiments, the effective daily dose of the active compound maybe administered as two, three, four, five, six, or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

In some embodiments, a compound as described herein is administeredalone. In some embodiments, a compound as described herein isadministered as a pharmaceutical formulation (composition) as describedherein.

In some embodiments, the compounds as described herein may be formulatedfor administration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

Preparation of Compounds and/or Compositions

In some embodiments, a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof, may beprepared in whole or in part by chemical synthesis; in some embodiments,a compound of any one of formulas (1)-(3), or a pharmaceuticallyacceptable salt, prodrug, or isomer thereof, prepared in part bychemical synthesis is prepared using semi-synthetic methodologies. Insome embodiments, a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof, may beprepared by isolation.

In some embodiments, the present disclosure provides methods forpreparing a Compound and/or Composition as described herein, comprisingassaying one or more samples, for example to detect bioactivity therein.In some embodiments, one or more of the samples comprise a compound ofany one of formulas (1)-(3), or a pharmaceutically acceptable salt,prodrug, or isomer thereof. In some embodiments, methods provided hereincomprise a step of detecting and/or confirming presence of detectablebioactivity in one or more samples. In some embodiments, methodsprovided herein comprise a step of confirming absence of detectablebioactivity in one or more samples.

In some embodiments, the bioactivity is inhibition of glucoseproduction. In some embodiments, the bioactivity is tested in HepG2cells. In some embodiments, the bioactivity is tested in H4IIE cells.

In some embodiments, the bioactivity is reduction of serum HbA1c level.In some embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

In some embodiments, the bioactivity is enhanced glucose tolerance. Insome embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

In some embodiments, the bioactivity is inhibition of G6pc expression.In some embodiments, the bioactivity is tested in AML-12 cells. In someembodiments, the bioactivity is tested in AML-12 cells stimulated withdiabetic stimuli. In some embodiments, the bioactivity is tested inhuman HepG2 cells. In some embodiments, the bioactivity is tested in theliver of insulin-resistant and diabetic mice.

In some embodiments, the bioactivity is enhanced phosphorylation ofPdk1, Akt, Foxo1 and AS160. In some embodiments, the bioactivity istested in the liver. In some embodiments, the bioactivity is tested inthe skeletal muscle.

In some embodiments, the bioactivity is enhanced Glut4 expression. Insome embodiments, the bioactivity is tested in mouse liver AML-12 cells.In some embodiments, the bioactivity is tested in the liver ofinsulin-resistant and diabetic mice.

In some embodiments, the bioactivity is activation and/or restoration ofinsulin signaling in a subject in insulin-resistant state. In someembodiments, the subject is characterized by significant levels ofcirculating insulin. In some embodiments, the insulin-resistant state ischaracterized by reduced level and/or activity of phosphorylated insulinreceptor in the subject. In some embodiments, the subject has diabetes,and/or diabetes associated disease, disorders, or conditions.

In some embodiments, the bioactivity is enhanced glucose uptake. In someembodiments, the cells are liver cells and skeletal muscle cells. Insome embodiments, the bioactivity is reduction of serum insulin level.In some embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

Identification and/or Characterization of Compounds and/or Compositions

In some embodiments, the present disclosure provides methods foridentifying and/or characterizing a compound and/or a composition asdescribed herein. In some embodiments, such a method comprises steps oftesting a plurality of samples, each of which comprises a compound ofany one of formulas (1)-(3), or a pharmaceutically acceptable salt,prodrug, or isomer thereof, for bioactivity therein; and determiningpresence and/or level of said bioactivity in one or more such samples.In some embodiments, a provided method comprises detecting saidbioactivity associated with presence and/or level of a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof. In some embodiments, a provided method comprises astep of identifying and/or characterizing a particular compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof, by detecting said bioactivity of the compound.

In some embodiments, the bioactivity is inhibition of glucoseproduction. In some embodiments, the bioactivity is tested in HepG2cells. In some embodiments, the bioactivity is tested in H4IIE cells.

In some embodiments, the bioactivity is reduction of serum HbA1c level.In some embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

In some embodiments, the bioactivity is enhanced glucose tolerance. Insome embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

In some embodiments, the bioactivity is inhibition of G6pc expression.In some embodiments, the bioactivity is tested in AML-12 cells. In someembodiments, the bioactivity is tested in AML-12 cells stimulated withdiabetic stimuli. In some embodiments, the bioactivity is tested inhuman HepG2 cells. In some embodiments, the bioactivity is tested in theliver of insulin-resistant and diabetic mice.

In some embodiments, the bioactivity is enhanced phosphorylation ofPdk1, Akt, Foxo1, and AS160. In some embodiments, the bioactivity istested in the liver. In some embodiments, the bioactivity is tested inthe skeletal muscle.

In some embodiments, the bioactivity is enhanced Glut4 expression. Insome embodiments, the bioactivity is tested in mouse liver AML-12 cells.In some embodiments, the bioactivity is tested in the liver ofinsulin-resistant and diabetic mice.

In some embodiments, the bioactivity is activation and/or restoration ofinsulin signaling in a subject in insulin-resistant state. In someembodiments, the subject is characterized by significant levels ofcirculating insulin. In some embodiments, the insulin-resistant state ischaracterized by reduced level and/or activity of phosphorylated insulinreceptor in the subject. In some embodiments, the subject has diabetes,and/or diabetes associated disease, disorders, or conditions.

In some embodiments, the bioactivity is enhanced glucose uptake intocells in a subject. In some embodiments, the bioactivity is enhancedglucose uptake into liver cells and skeletal muscle cells. In someembodiments, the bioactivity is reduction of serum insulin level. Insome embodiments, the bioactivity is tested in insulin-resistant anddiabetic mice.

Uses

The present disclosure provides that the compounds as described herein,for example compound 43, can mimic insulin to inhibit glucose production(see for example, Example 2); lower blood glucose and HbA1c levels,attenuate the development of hyperglycemia and improve glucose tolerancein insulin-resistant diabetic subjects (see for example, Example 3);inhibit G6pc expression in the liver of insulin-resistant diabeticsubjects, and mimic but bypass insulin to inhibit G6pc expression incultured mouse and human liver cells and potentiate insulin action (seefor example, Example 4); mimic but bypass insulin to activate Pdk1 andAkt and enhance Foxo1 phosphorylation in the liver (see for example,Example 5); enhance Glut4 expression in the liver of insulin-resistantdiabetic subjects, and mimic but bypass insulin to enhance Glut4expression in mouse liver cells (see for example, Example 6), thephosphorylation of AS160 (a key event for GLUT4 transportation fromcytosolic vesicles to plasma membrane to facilitate glucose uptake) inhuman liver cells (see for example, Example 8), and glucose uptake inmouse liver cells (see for example, Example 6); enhance and/or restoreinsulin signaling Pdk1/Akt/Foxo1 in the skeletal muscles ofinsulin-resistant diabetic subjects (see for example, Example 7), mimicby bypass insulin to activate Akt and enhance the phosphorylation ofAS160 (a key event for GLUT4 transportation from cytosolic vesicles toplasma membrane) in skeletal muscle cells (see for example, Example 8),and potentiate insulin action to stimulate glucose uptake in skeletalmuscle cells (see for example, Example 7); activate and/or restoreinsulin receptor (Insr) function in the skeletal muscle and liver ofinsulin-resistant diabetic subjects and in cultured mouse skeletalmuscle and human liver cells (see for example, Example 8); and attenuatehyperinsulinemia without impairing kidney function and/or resulting inliver damage (Example 9). Accordingly, the present disclosure clearlydemonstrates that the compounds as described herein are useful formodulating glucose metabolism and treating an insulin-related disorderas described herein.

In some embodiments, the present disclosure provides methods formodulating glucose metabolism and/or treating glucose metabolismdisorders, comprising administering a therapeutically effective amountof a compound of any one of formulas (1)-(3), or a pharmaceuticallyacceptable salt, prodrug, or isomer thereof. In some embodiments,glucose metabolism disorders involve a blood glucose level which is notwithin the normal range. In some embodiments, glucose metabolismdisorders relate to defective glucose uptake and/or transport. In someembodiments, glucose metabolism disorders are Diabetes Mellitus,glyceraldehyde-3-phosphate dehydrogenase deficiency, glycosuria,hyperglycemia, hyperinsulinism, or hypoglycemia.

In some embodiments, the present disclosure provides methods fortreating disorders of glucose transport, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, disorders of glucose transport areglucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivodisease (GLUT1 deficiency syndrome (GLUT1DS)).

In some embodiments, the present disclosure provides methods forenhancing AS160 phosphorylation for translocation of glucose transporterproteins (GLUTs) from cytosolic vesicles to plasma membrane for glucoseuptake, comprising administering a therapeutically effective amount of acompound of any one of formulas (1)-(3), or a pharmaceuticallyacceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forenhancing glucose uptake in both liver and skeletal muscles, comprisingadministering a therapeutically effective amount of a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating an insulin-related disorder, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, the insulin-related disorders are selectedfrom the group consisting of pre-diabetes, type I diabetes, type IIdiabetes, hypoglycemia, hyperglycemia, insulin resistance, secretorydysfunction, loss of pancreatic β-cell function, and loss of pancreaticβ-cells. In some embodiments, the patients of insulin-related disordersare non-insulin dependent patients having insulin-related disorders.

In some embodiments, the present disclosure provides methods fortreating insulin resistance disorder comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. Certain examples of insulin resistance disorders are describedabove. In some embodiments, provided methods are for treating Type IIdiabetes, hyperinsulinemia, hyperproinsulinemia, retinopathy,neuropathy, or nephropathy. In some embodiments, provided methodsattenuate hyperinsulinemia without impairing kidney function and/orresulting in liver damage.

In some embodiments, the present disclosure provides methods fortreating obesity comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods fortreating diabetes, comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, the diabetes is type I diabetes. In some embodiments, thediabetes is type II diabetes.

In some embodiments, the present disclosure provides methods fortreating hyperglycemia comprising administering a therapeuticallyeffective amount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forinhibiting glucose production, comprising administering a compound ofany one of formulas (1)-(3), or a pharmaceutically acceptable salt,prodrug, or isomer thereof.

In some embodiments, the present disclosure provides methods forreducing serum HbA1c level, comprising administering a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods forincreasing glucose tolerance, comprising administering a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods forinhibiting G6pc expression, comprising administering a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods forenhancing phosphorylation of Pdk1, Akt, and Foxo1 in the liver and/or inthe skeletal muscle, comprising administering a compound of any one offormulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, orisomer thereof.

In some embodiments, the present disclosure provides methods forincreasing Glut4 expression, comprising administering a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, the present disclosure provides methods foractivating and/or restoring insulin signaling in a subject ininsulin-resistant state, comprising administering a compound of any oneof formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug, orisomer thereof. In some embodiments, the subject is characterized bysignificant levels of circulating insulin. In some embodiments, theinsulin-resistant state is characterized by reduced level and/oractivity of phosphorylated insulin receptor in the subject. In someembodiments, the subject has diabetes, and/or diabetes associateddisease, disorders or conditions. In some embodiments, the subject hastype II diabetes.

In some embodiments, the present disclosure provides methods forenhancing glucose uptake into cells in a subject, comprisingadministering a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. In someembodiments, the cells are skeletal muscle cells.

It has been shown that Compound #43 can mimic but bypass insulin toactivate insulin receptor signaling in the liver and skeletal muscle,and can restore insulin receptor function, even in insulin-resistantdiabetic subjects. These observations suggests that compound #43 can beused for the treatment of diseases or syndromes characterized bydefective insulin signaling, such as polycystic ovary syndrome (PCOS),Alzheimer's disease (AD) and sarcopenia.

In some embodiments, the present disclosure provides methods fortreating polycystic ovary syndrome (PCOS) comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. PCOS is a hormone imbalance that can cause irregular periods,unwanted hair growth, and acne. Young women with PCOS often haveelevated insulin levels which can cause the ovaries to make moreandrogen hormones, resulting in increased body hair, acne, and irregularor few periods. Having PCOS can cause insulin resistance and thedevelopment of type 2 diabetes. Metformin is a medication oftenprescribed for women with PCOS to improve insulin sensitivity andprevent the development of type 2 diabetes. The results demonstrate thatcompound #43 is more potent than metformin in the inhibition of glucoseproduction in cultured liver cells, and can restore insulin receptorfunction in insulin-resistant diabetic mice. Thus, Compound #43 can bepotentially useful for the treatment of PCOS.

In some embodiments, the present disclosure provides methods fortreating Alzheimer's disease (AD) comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. Brain insulin signaling is important for learning and memory,and insulin resistance in the brain is a major risk factor for AD. Therestoration of insulin signaling has emerged as a potential therapy forAD (White M F, Science 2003; 302:1710-1; De Felice D G et al,Alzheimer's & Dementia 2014; 10: S26-S32). In view of the results aboveshowing that Compound #43 exhibited insulin-like activity and was ableto restore insulin receptor function in insulin-resistant subjects, itcan be potentially useful for the treatment of AD.

In some embodiments, the present disclosure provides methods fortreating sarcopenia comprising administering a therapeutically effectiveamount of a compound of any one of formulas (1)-(3), or apharmaceutically acceptable salt, prodrug, or isomer thereof. Sarcopeniais characterized by the progressive loss of skeletal muscle mass withincreasing age, leading to decreased muscle strength, decreased mobilityand function, increased fatigue, an elevated risk of fall-relatedinjury, and, often, frailty (Candow and Chilibeck, 2005; Sakuma andYamaguchi, 2012). Recent advances in muscle biology have revealed thatinsulin signaling (Insr/PI3K/Akt) is critical for the synthesis ofmuscle protein, and the inhibition of muscle protein degradation(through Akt/Foxo1-mediated inhibition of the expression of two atrophygenes Fbxo32 and Trim63). Optimal insulin signaling attenuates musclewasting processes, including sarcopenia (Glass and Roubenoff, 2010;Ryall et al., 2008; Sakuma and Yamaguchi, 2012). As such, stimulators ofinsulin signaling, amino acid supplements (to improve protein synthesis)and inhibitors of proteasome protein degradation are emerging as newstrategies for the treatments of sarcopenia. The insulin-like activityof Compound #43 in the activation of Insr/Pdk1/Akt/Foxo1 signaling inskeletal muscle even in insulin-resistant diabetic mice, as describedherein, suggests the potential use of this compound for treatingatrophic conditions in muscles.

The present disclosure also teaches that provided compounds likelyenhance mitochondrial function, and are therefore useful for treatingmitochondrial diseases and/or dysfunction. In some embodiments, thepresent disclosure provides methods for treating mitochondria-associateddiseases (e.g., caused by dysfunctional mitochondria), comprisingadministering a therapeutically effective amount of a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof. In some embodiments, mitochondria-associated diseasescan be degenerative diseases (e.g., cancer, cardiovascular disease andcardiac failure, type 2 diabetes, Alzheimer's and Parkinson's diseases,fatty liver disease, cataracts, osteoporosis, muscle wasting, sleepdisorders and inflammatory diseases such as psoriasis, arthritis andcolitis). In some embodiments, the present disclosure provides methodsfor enhancing mitochondrial function, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof.

In some embodiments, the present disclosure provides methods forenhancing gluconeogenesis in the brain, comprising administering atherapeutically effective amount of a compound of any one of formulas(1)-(3), or a pharmaceutically acceptable salt, prodrug, or isomerthereof. In some embodiments, provided methods increase glucose uptakein the brain. In some embodiments, provided methods are for maintainingor restoring brain functions including memory and learning.

Combination Therapy

In some embodiments, the present disclosure provides use of thecompounds and/or compositions as described herein for combinationtherapy of a disease, disorder, or condition as described herein. Insome embodiments, the present disclosure provides methods for treatingpatients with a disease, disorder, or condition, who have received, arereceiving, or will receive one or more different therapies for saiddisease, disorder, or condition, wherein the methods compriseadministering a therapeutically effective amount of a compound of anyone of formulas (1)-(3), or a pharmaceutically acceptable salt, prodrug,or isomer thereof.

In some embodiments, one or more therapies for patients withinsulin-related disorders are selected from the group consisting ofinsulin therapy, for example, for type I diabetes; diet and exercise,for example, for incipient type II diabetes; oral antidiabetic agents,for example, for early stage type II diabetes; metformin; insulinsecretagogues, for example, sulfonylureas; glitazones; long-acting basalinsulin; intermediate acting insulin; and short (rapid) acting insulin.In some embodiments, insulin therapy involves administrationsubcutaneously (SC), intravenously, and/or by inhalation. In someembodiments, one or more therapies for patients with insulin-relateddisorders may be therapies currently under development, for example,insulin-fumaryl diketopiperazine (FDKP).

EXAMPLES Example 1: Synthesis of Compounds #43, #50, #53, #69, #70, and#68 1a. Synthesis of Adenosine, 5′-Se-methyl-5′-selino-, 2′,3′-diacetate(Compound #43)

Synthetic procedure: 5′-Se-methyl-5′-seleno-Adenosine (1.0 gr, 0.0029mole, 1.0 mole eq.) and anhydrous pyridine (10 ml) were placed in anoven dried, 50 ml three neck flask, equipped with a dropping funnel,inert gas inlet/outlet and a thermometer. The reaction set was placed inan ice/salt bath and agitation was initiated. When the temperature ofthe solution dropped to 0° C., acetic anhydride (10 ml, 0.105 mole,36.47 mole eq.) was added drop-wise for 15 minutes and the temperatureof the reaction mixture was maintained below 5° C. during aceticanhydride addition. The reaction mixture was stirred for 6 hours at5-10° C. Quenched the excess acetic anhydride by adding ice-cold water(100 ml), and then pH adjusted to 7 by adding 10 wt % NaHCO₃ aqueoussolution. The aqueous mixture was extracted with ethyl acetate (2×100ml). The combined ethyl acetate extracts are dried over anhydrous Na₂SO₄(1 gr), filtered into a 250 ml round-bottomed flask. Concentrated thefiltrate to dryness under reduced pressure at 35-40° C. to give thecrude product as a pale yellow syrupy liquid, then pure product wasobtained as off-white crystals (1.12 gr, Yield: 90.3%, Purity byHPLC: >99%) by passing through a silica gel column with a mixture ofethyl acetate and hexanes (1:3 v/v).

1b. Synthesis of Adenosine, 5′-S-methyl-5′-thio-, 2′,3′-diacetate(Compound #68)

Synthetic procedure: 5′-S-methyl-5′-thio-Adenosine (1.0 gr, 0.0033 mole,1.0 mole eq.) and anhydrous pyridine (10 ml) were placed in an ovendried, 50 ml three neck flask, equipped with a dropping funnel, inertgas inlet/outlet and a thermometer. The reaction set was placed in anice/salt bath and agitation was initiated. When the temperature of thesolution dropped to 0° C., acetic anhydride (10 ml, 0.105 mole, 31.8mole eq.) was added drop-wise for 15 minutes and the temperature of thereaction mixture was maintained below 5° C. during acetic anhydrideaddition. The reaction mixture was stirred for 6 hours at 5-10° C.Quenched the excess acetic anhydride by adding ice-cold water (100 ml),and then pH adjusted to 7 by adding 10 wt % NaHCO₃ aqueous solution. Theaqueous mixture was extracted with ethyl acetate (2×100 ml). Thecombined ethyl acetate extracts are dried over anhydrous Na₂SO₄ (1 gr),filtered into a 250 ml round-bottomed flask. Concentrated the filtrateto dryness under reduced pressure at 35-40° C. to give the crude productas a pale yellow syrupy liquid, then pure product was obtained asoff-white crystals (1.08 gr, Yield: 87%, Purity by HPLC: >99%) bypassing through a silica gel column with a mixture of ethyl acetate andhexanes (1:3 v/v).

1c. Synthesis of Adenosine, 5′-Se-methyl-5′-selino-, 2′,3′-dipropionate(Compound #69)

Synthetic procedure: 5′-Se-methyl-5′-seleno-Adenosine (1.0 gr, 0.0029mole, 1.0 mole eq.) and anhydrous pyridine (10 ml) were placed in anoven dried, 50 ml three neck flask, equipped with a dropping funnel,inert gas inlet/outlet and a thermometer. The reaction set was placed inan ice/salt bath and agitation was initiated. When the temperature ofthe solution dropped to 0° C., propionic anhydride (10 ml, 0.078 mole,27.0 mole eq.) was added drop-wise for 15 minutes and the temperature ofthe reaction mixture was maintained below 5° C. during propionicanhydride addition. The reaction mixture was stirred for 6 hours at5-10° C. Quenched the excess propionic anhydride by adding ice-coldwater (100 ml), and then pH adjusted to 7 by adding 10 wt % NaHCO₃aqueous solution. The aqueous mixture was extracted with ethyl acetate(2×100 ml). The combined ethyl acetate extracts are dried over anhydrousNa₂SO₄ (1 gr), filtered into a 250 ml round-bottomed flask. Concentratedthe filtrate to dryness under reduced pressure at 35-40° C. to give thecrude product as a pale yellow syrupy liquid, then pure product wasobtained as off-white crystals (1.18 gr, Yield: 89.3%, Purity byHPLC: >99%) by passing through a silica gel column with a mixture ofethyl acetate and hexanes (1:3 v/v).

1d. Synthesis of Adenosine, 5′-Se-methyl-5′-selino-, 2′,3′-dibutanoate(Compound #70)

Synthetic procedure: 5′-Se-methyl-5′-seleno-Adenosine (1.0 gr, 0.0029mole, 1.0 mole eq.) and anhydrous pyridine (10 ml) were placed in anoven dried, 50 ml three neck flask, equipped with a dropping funnel,inert gas inlet/outlet and a thermometer. The reaction set was placed inan ice/salt bath and agitation was initiated. When the temperature ofthe solution dropped to 0° C., butyric anhydride (10 ml, 0.078 mole,27.0 mole eq.) was added drop-wise for 15 minutes and the temperature ofthe reaction mixture was maintained below 5° C. during butyric anhydrideaddition. The reaction mixture was stirred for 6 hours at 5-10° C.Quenched the excess butyric anhydride by adding ice-cold water (100 ml),and then pH adjusted to 7 by adding 10 wt % NaHCO₃ aqueous solution. Theaqueous mixture was extracted with ethyl acetate (2×100 ml). Thecombined ethyl acetate extracts are dried over anhydrous Na₂SO₄ (1 gr),filtered into a 250 ml round-bottomed flask. Concentrated the filtrateto dryness under reduced pressure at 35-40° C. to give the crude productas a pale yellow syrupy liquid, then pure product was obtained asoff-white crystals (1.20 gr, Yield: 85.7%, Purity by HPLC: >99%) bypassing through a silica gel column with a mixture of ethyl acetate andhexanes (1:3 v/v).

1e. Synthesis of Adenosine, 5′-Se-methyl-5′-seleno-, cyclic2′,3′-carbonate (Compound #50)

Synthetic procedure: 5′-Se-methyl-5′-seleno-Adenosine (1.0 gr, 0.0029mole, 1.0 mole eq.) and anhydrous dimethylformamide (20 ml) were placedin an oven dried, 50 ml three neck flask, equipped with a droppingfunnel, inert gas inlet/outlet and a thermometer. The reaction set wasplaced in an ice/salt bath and agitation was initiated. When thetemperature of the solution dropped to 0° C., carbonyldiimidazole (CDI,0.57 gr, 0.0035 mole, 1.21 mole eq.) was added at below 5° C. Thereaction mixture was slowed warmed to the room temperature, and thenstirred the reaction mixture for 4 hours at the same temperature underargon gas atmosphere. The solvent was removed under reduced pressure togive the residue, it was dissolved in a mixture of chloroform (5 ml) andethanol (few drops) to get clear solution, washed the organic layer with1% aq. acetic acid solution (2×1 ml), dried over anhydrous Na₂SO₄ (1gr), filtered into a 250 ml round-bottomed flask. Concentrated thefiltrate to dryness under reduced pressure at 25-30° C. to give thecrude product as a pale yellow syrupy liquid. Dissolved the crudeproduct in a mixture of ethanol/water mixture (1:1 v/v), and thenconcentrated to dryness under reduced pressure at 45-50° C. to give aresidue, hexanes (25 ml) were added and stirred for 10 minutes, and thenconcentrated to dryness under reduced pressure at 30-35° C. to yield thedesired product as a off-white crystals (1.02 gr, Yield: 95.3%, Purityby HPLC: >99%).

1f. Synthesis of a Region-Isomeric Mixture of Adenosine,5′-Se-methyl-5′-seleno-, 2′-morpholinocarbamate and Adenosine,5′-Se-methyl-5′-seleno-, 3′-morpholinocarbamate (Compound #53)

Synthetic procedure: Adenosine, 5′-Se-methyl-5′-seleno-, cyclic2′,3′-carbonate (1.0 gr, 0.0027 mole, 1.0 mole eq.) and anhydrousdimethylformamide (10 ml) were placed in an oven dried, 50 ml three neckflask, equipped with a dropping funnel, inert gas inlet/outlet and athermometer. Morpholine (0.26 gr, 0.0029 mole, 1.1 mole eq.) was addedat 20-25° C. Stirred the reaction mixture for 1 hour at roomtemperature, and then concentrated to dryness under reduced pressure at45-50° C. to give a residue, hexanes (25 ml) were added and stirred for10 minutes to precipitate the desired region-isomeric mixture product asa off-white solid (0.12 gr, Yield: 91%, Purity by HPLC: >99%).

Example 2

Synthetic compounds listed in Table 1 were tested in cell culture (invitro) for effects on glucose production and cell viability in theexamples described herein. In particular, the cells tested were humanhepatoma HepG2 and rat liver H4IIE liver cells.

TABLE 1 Abbreviated compound Mol. name Compound name Compound structureWt. Solvent Compound-C 5′- Methylselenoadenosine

344.22 DMSO Compound-D 5′- Selenoadenosyl- homocysteine

431.30 DMSO Compound-E γ-L-glutamyl-Se- methyl-L- selenocysteine

311.19 Water Compound #43 (Diacetyl ester of Compound-C) Diacetyl esterof methyl selenoadenosine

428.30 DMSO Compound #50 Cyclic carbonate of methylselenoadenosine

370.22 DMSO Compound #53 Carbamate analog of methylselenoadenosine withmorpholine

457.34 DMSO

Compound #57 Adenosine

267.24 DMSO Compound #59 (a cell- permeable adenylyl cyclase inhibitor)2′,5′- Dideoxyadenosine

235.24 DMSO Compound #60 5′-Deoxyadenosine

251.24 Water Compound #61 (an adenylyl cyclase inhibitor)9-(tetrahydrofuran-2- yl)-9h-purin-6-amine (SQ 22,536)

205.21 DMSO Compound #62 Adenine

135.12 DMSO Compound #63 Nicotinamide adenine dinucleotide (NAD)

664.43 Water Compound #64 S-(5′-Adenosyl)-L- methionine iodide (SAM)

526.35 Water Compound #68 (Sulfur analog of Compound #43) Diacetyl esterof methylthiooadenosine

381.40 DMSO Compound #69 (Dipropanoyl ester of Compound-C) Dipropanoylester of methylselenoadenosine

456.35 DMSO Compound #70 (Dibutanoyl ester of Compound-C) Dibutanoylester of methylselenoadenosine

484.40 DMSOMaterials and MethodsCell Lines and Compounds

The human hepatoma HepG2 and rat hepatoma H4IIE cells were purchasedfrom the American Type Culture Collection (ATCC, Manassas, Va.). HepG2cells and H4IIE cells were cultured in Eagle's Minimum Essential Medium(EMEM) supplemented with 10% FBS.

Compound #43 and other compounds listed in Table 1 were eithersynthesized or obtained from commercial sources (where available). Thepurities of all tested compounds were verified to be ≥99%, as determinedby HPLC. All these compounds were either dissolved in DMSO or in waterto obtain a 126.7 or 12.67 mM stock solution for experiments. Metforminand insulin were purchased from Sigma.

Glucose Production Assay

Equal numbers of Human HepG2 or rat H4IIE cells (1-1.5×10⁵ cells/well)were seeded on 96 well plates in 10% FBS-containing media for 24 hr.Cells were then washed twice with PBS, and treated with variousconcentrations of compounds, metformin or insulin in 100 μl of glucoseproduction media (glucose-free, phenol red-free DMEM media supplementedwith 20 mM sodium lactate, 2 mM sodium pyruvate and 5 mM HEPES) at 37°C. for 24 hr (H4IIE cells only) or 48 hr (HepG2 cell only). Cells werealso incubated with 0.24% DMSO (the maximal volume of DMSO solvent usedin the experiments).

After the above treatments, 50 μl of culture media were collected, andsubjected to glucose analysis using Molecular Probes Amplex Red glucoseassay kit (Cat #A22189) according to the manufacturer's protocol. Cellnumbers or viability in the culture plate after the above compoundtreatments were determined using Promega's CellTiter96® AQueous OneSolution Cell Proliferation Assay kits, according to the manufacturer'sprotocol and instructions. In brief, after removing 50 μl of culturemedia for glucose assay, cells were incubated with AQueous One solution(25 μl stock solution and 50 μl prewarmed PBS/per well) at 37° C. for 1hour, and the absorbance of OD490 nm in each sample was determined bythe Bio-Tek microplate reader. Cell viability in culture wells weredetermined by the subtraction of OD490 nm in cultured cells with theOD490 nm in plain culture media (without seeding of cells). Glucoseproduction in the culture cells were obtained by normalizing the glucoseconcentration in culture media by cell viability in each well. At least3 samples per each treatment were examined for the above analysis. Dataare presented as Mean±SEM of those samples. Experiments were repeated atleast twice.

The half maximal inhibitory concentration (IC50 values) of Compound #43or metformin for the inhibition of glucose production were determinedusing the ED50 Plus v1.0 online software.

Results and Discussion

1. Effect of Insulin and Compound Solvent DMSO on Glucose Production inHepG2 Cells

As shown in the left panel of FIG. 1, treatment with 10 nM and 100 nMinsulin, respectively, resulted in a 20% and 30% reduction in glucoseproduction in HepG2 cells while the compound solvent DMSO at the maximalvolume used did not affect glucose levels in cultured HepG2 cells. Theseresults suggest that HepG2 cells are responsive to insulin in theinhibition of glucose production, and the observed effects of the testedcompounds on glucose production are not due to potential effects ofDMSO. These results establish that HepG2 cells constitute an appropriatecell system for the screening of compounds having insulin-like activityand which can inhibit glucose production in liver cells.

2. Compound #43 can Mimic Insulin to Inhibit Glucose Production in HepG2Cells

As shown in FIG. 1, incubation of HepG2 cells with Compound #43 at thetested doses under serum-free conditions resulted in a decrease inglucose levels in the culture media. The observed reduction in glucoseproduction by HepG2 cells after treatment with compound #43 (3.8 μM) wascomparable to that achieved when insulin (100 nM) was used, while higherdoses of Compound #43 (7.6, 15.2 and 30.4 μM) were much more potent than100 nM insulin. Furthermore, no significant decrease of cell viabilitywas observed in the HepG2 cells after the treatment of Compound #43 atall tested doses (data not shown). These results suggest that Compound#43 is an insulin-mimetic that can inhibit glucose production in HepG2cells.

3. Compound #43 on its Own Showed Higher Endpoint Potency than the ThreeCompound Combination, CDE, in the Inhibition of Glucose Production inHepG2 Cells

Treatment of HepG2 cells with Compound CDE, at all tested doses, (1:1:1ratio of C/D/E) also inhibited glucose production with a potencycomparable to 100 nM insulin. Compound #43 at a dose of 3.8 μM was aspotent as the CDE combination product (which contained 3.8 μM of eachindividual compound) in inhibiting glucose production in HepG2 cells.However, Compound #43 at a dose of 7.6 μM or higher was more potent thanCDE in inhibiting glucose production in HepG2 cells. These resultsdemonstrated that Compound #43 on its own showed higher endpoint potencythan the three compound combination, CDE, in inhibiting glucoseproduction in HepG2 cells. Noting that the selenium concentration inCompound CDE is three times higher than in Compound #43 at each dosepoint, these results might suggest that one or more structural featuresof Compound #43 in addition to its selenium molecule may be able, atleast in certain circumstances, contribute to its activity.

4. Inhibition of Glucose Production in HepG2 is Abrogated when Sulfur isSubstituted for Selenium Molecule in Compound #43

As shown in the right-hand panel of FIG. 1, treatment of cells withsulfur-bearing compounds, Compound #68 (the sulfur analog of Compound#43) or Compound #64 (SAM), as well as other listed non-seleniumcompounds had minimal effects in lowering glucose production in HepG2cells. The striking difference in the inhibition of glucose productionbetween Compound #43 and #68 demonstrates that the selenium molecule inCompound #43 contributes to its function in the inhibition of glucoseproduction in HepG2 cells.

5. Differential Effect of Selenium Compounds on the Inhibition ofGlucose Production in HepG2 Cells

As described above, an equimolar mixture of the selenium-containingcompounds C, D, and E (CDE) inhibited glucose production in human HepG2cells at the indicated doses. Noting that the compound concentrationindicated on the X-axis of FIG. 1 refers to the Se-concentration of eachSe-containing compound in the mixture, the total Se-concentration isactually three-times higher than that is indicated on the X-axis. Thiswas done to facilitate direct comparison of the mixture components witheach of the single molecule candidates tested in those experiments.

If the inhibition of glucose production was solely due to selenium, thenit might be expected that CDE, because it contains three-times moreselenium than Compound #43, would produce a more robust response thanthe latter. That is clearly not the case.

FIG. 1 (the left-hand panel) shows that each of compounds C, D, 43, 50,53, 69 and 70 inhibited glucose production, among which compound 43 wasthe most potent. Further, compound E was found to stimulate glucoseproduction in HepG2 cells.

Taken together, these results suggests:

-   -   (i) The selenium molecule in the compound is required for        effective inhibition of glucose production (compare Compound #43        in the left-hand panel of FIG. 1 to its exact sulfur analog,        Compound #68 in the right-hand panel).    -   (ii) However, the selenium molecule alone may not be sufficient        for inhibiting glucose production (in some cases,        selenium-containing, structurally similar compounds exhibited        lower or opposite effect);    -   (iii) Increasing the concentration of selenium molecule alone        may not enhance potency (selenium concentration of CDE is        three-fold higher than that of Compound #43, yet is not as        potent);    -   (iv) Among the compounds tested, Compound #43 exhibited the        highest potency in inhibiting glucose production in liver cells.

6. Analysis of Structural Features of the Listed Selenium Compounds toDetermine Chemical Groups which May Contribute to their Activity

Comparison of Compounds C and D indicates that 5′ methyl seleno groupand 5′ seleno homocysteine may provide similar inhibition of glucoseproduction. Comparison of Compounds 43, 50, 53, 69, and 70 indicatesthat diacetyl ester at 2′ and 3′ position (#43) provides higherinhibition of glucose production than cyclic carbonate (Compound #50),morpholino carboxylate (#53), dipropanoyl ester (#69), and dibutanoylester (#70). Adenine, adenosine and several chemical variants ofadenosine did not inhibit glucose production in HepG2 cells.Furthermore, two adenylyl cyclase inhibitors (#59 and 61) also did notinhibit glucose production, indicating that the action of Compound #43in the inhibition of glucose production is unlikely due to a decrease incellular AMP levels (Hardie D G. Cell Metabolism 2013: 17(3): 313-314).Together, the results suggest that one or more features, in addition toadenine and selenium molecule, may be able, at least in certaincircumstances, to contribute to the activity.

7. Comparative Studies of Compound #43 and Metformin (a Well-KnownAntidiabetic Drug) in the Inhibition of Glucose Production in HepG2 andRat H4IIE Cells

As described above, the robust inhibition of glucose production in HepG2cells was observed after Compound #43 treatment. The effects of thiscompound were compared to the well-known anti-diabetic drug metformin.As described above and shown in the top panel of FIG. 2, adose-dependent decrease of glucose levels in HepG2 cells followingCompound #43 treatment was observed and the IC50 (the half maximalinhibitory concentration) value was 15.5 μM. As can be seen, metforminat a dose of 500 μM did not inhibit glucose production in HepG2 cellsbut instead showed some stimulatory effects. Higher doses of metformin(0.5-4 mM) showed cell toxicity on these cells (data not shown). Theseresults demonstrate that Compound #43, similar to insulin, can inhibitglucose production in HepG2 cells.

It has been reported that rat H4IIE liver cells can respond to metforminleading to lowered glucose production. Thus, this rat liver cell linewas used to further confirm the inhibitory activity of Compound #43 onglucose production, and to compare the potency of Compound #43 withmetformin. As shown in FIG. 2, treatment with Compound #43 andmetformin, respectively, resulted in a dose-dependent decrease inglucose production in H4IIE cells under serum-free conditions. No toxiceffect of these compounds was observed at the tested doses on cellviability (data not shown). The IC50 of Compound #43 was 17.8 μM, whichis nearly identical to its IC50 in HepG2 cells. In contrast, metforminat a dose of 36.25 μM showed little or no inhibitory activity, and theIC50 of metformin in this experiment was 275 μM. These results suggestthat Compound #43 is more potent (at least 15 times more potent) thanmetformin in the inhibition of glucose production in cultured rat livercells.

In summary, Compounds #43, 50, 53, 69 and 70 all displayed activity inthe inhibition of glucose production in cultured liver cells. However,Compound #43 was by far the most potent single compound tested with aninhibitor activity against glucose production which exceeded that of ahigh insulin dose (100 nM) in HepG2 cells.

Moreover, Compound #43 was demonstrated to be much more potent than thebiguanide drug, metformin, in both liver cell lines tested. Metformincurrently is the first-line drug used in the treatment of type 2diabetes.

Example 3: Studies with Compound #43 and the Closely RelatedSeleno-Organic Compounds (#C, #50, #69 and #70), Together with theSulfur Analog of Compound #43 (Compound #68) in the Regulation ofGlucose and/or HbA1c Levels in the Bloodstream and in the Improvement ofGlucose Tolerance in Insulin-Resistant and Diabetic Leptin Receptor(Lepr) Spontaneous Null Mutant Mice

Materials and Methods

Compounds

Compound #43, #C, #50, #68, #69 and #70 were synthesized in theChemistry Laboratory of Alltech, Inc. The purities of all testedcompounds were verified to be ≥99%, as determined by HPLC.

Animals

5-10 week-old male diabetic spontaneous mutation (leptin receptormutation) Lepr^(db/db) mice (C57BL/6J strain) were purchased from TheJackson Laboratory (Bar Harbor, Me.), and housed in a pathogen-freevivarium with free access to chow and water.

Chronic Treatments with Compound #43 and Other Compounds

Male Lepr^(db/db) mice at 38 days of age were intraperitoneally (ip)injected daily with physiological saline (0.09% NaCl) containing 0.2%DMSO, Compound #C, Compound #50, Compound #68, and/or Compound #43 (25μg selenium or sulfur equivalents of each compound per kilogram bodyweight, diluted in sterile physiological saline) for periods rangingfrom 43-90 days. Body weights of the above treated mice were recordeddaily using a balance and any visible abnormal animal gross morphologyand walking behavior were monitored daily. After the treatments, animalswere fasted overnight and then subjected to blood glucose or HbA1cassays, glucose tolerance tests or tissue collections.

For comparative studies of any potential anti-diabetic effects ofCompounds #43, #69 and #70, male Lepr^(db/db) mice at 41 days of agewere intraperitoneally (ip) injected daily with physiological saline(0.09% NaCl) containing 0.2% DMSO, Compound #43, Compound #69, orCompound #70 (25 μg selenium of each compound per kilogram body weight,diluted in sterile physiological saline). After the treatments for 43days, animals were fasted overnight and then subjected to blood glucoseand glucose tolerance tests. After daily treatments of these compoundsfor 90 days, sera from these animals were collected and then subjectedto blood HbA1c assays.

Acute Treatment of Compound #43

After overnight fasting, 8-10-week-old Lepr^(db/db) mice wereintraperitoneally injected with physiological saline (0.09% NaCl)containing 0.2% DMSO, or Compound #43 (0.0054, 0.054, 0.54, or 5.4 mgCompound #43 (the compound stock was diluted in sterile physiologicalsaline) per kilogram of body weight. Following the injection of salineor compound, mice were returned to their cages with free access to waterbut not chow for 1, 2, 3, 5 and 8 hr. At each time point afterinjection, a small drop of blood was collected from the tail of eachmouse for glucose assay.

Additional 6-week old Lepr^(db/db) mice under non-fasting conditionswere intraperitoneally injected with physiological saline (0.09% NaCl)containing 0.2% DMSO, or a single dose of Compound #43 (5.4 mgcompound/kg body weight). After injection, animals were returned back totheir cages with free access to water and chow. After 24 hr, a smalldrop of blood from the mouse tail was collected for glucose assay.

Blood Glucose Assay

After treatment with physiological saline or compounds or after theinjection of a bolus of glucose, a small drop of blood from each mousewas collected by snipping the mouse tail tip. Blood glucose levels weredetermined using a Glucometer with a maximum capability for glucosemeasurement of 600 mg/dL.

Glucose Tolerance Test

Glucose tolerance tests were performed as described previously (Li etal, Int J Biol Sci 2008; 4:29-36). Briefly, overnight-fastedLepr^(db/db) mice after saline or compound treatments were injectedintraperitoneally with 2 grams/kg body weight of 20% D-glucose. Bloodglucose levels at time 0 (immediately before the injection of glucose),0.25, 0.5, 1 and 2 hours after injection of glucose were determinedusing a glucometer with a maximum glucose measurement capacity of 600mg/dL. Because of this, blood glucose levels over 600 mg/dL were countedas 600 mg/dL inthe data analysis.

Blood and Serum HbA1c Assays

After saline or compound treatments, a small drop of blood from themouse tail was collected in an EDTA-coated eppendorf tube (to preventblood coagulation) (Fisher Scientific), and then subjected to a HbA1cassay using the Crystal Chem's or Kamiya's mouse glycated hemoglobin A1cELISA kit, according to the manufacturer's protocol. Also after thefinal treatments, mouse serum was collected and subjected to HbA1c assayusing the Kamiya Biomedical Company's mouse HbA1c kit, according to themanufacturer's protocol.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of difference between saline- andcompound-treated groups, with a P value less than 0.05 being deemedsignificant. Data are presented as mean±SEM of the indicated numbers ofmice in the figures.

Results and Discussion

Lepr^(db/db) mice lack all known isoforms of the leptin receptor gene(Lepr). This homozygous mouse model is an aggressive Type II diabeticmouse model with impaired glucose tolerance, reduced insulinsensitivity, hyperglycemia and hyperinsulinemia. These mice displaygross obesity at around 3 to 4 weeks of age, elevation of plasma insulinbeginning at 10 to 14 days and hyperglycemia (i.e., high blood sugarlevels) developed at about 4-8 weeks of age (Coleman D L. 1978Diabetologia 14:141-8).

In vitro studies showed that Compound #43 can mimic but bypass insulinto inhibit glucose production with a much greater potency than closelyrelated compounds such as Compound #C or #50. Furthermore, thesulfur-containing analog of Compound #43 (Compound #68) had little or noinhibitory effect on glucose production in HepG2 cells (FIGS. 1-2).Therefore, the insulin-resistant Lepr^(db/db) mouse is an ideal in-vivomodel system to investigate the use of experimental compounds inpotentially lowering glucose in the bloodstream and improving insulinsensitivity and glucose tolerance against a severe diabetic background.

1. Compound #43, the Most Potent Compound Among Three TestedSeleno-Organic Compounds (#C, #50, #43) Against Hyperglycemia inLepr^(db/db) Mice after Chronic Treatments

The administration regimes of compounds were adopted to investigate thepotential role of test compounds in the treatment of hyperglycemia, asdisplayed in Lepr^(db/db) mice. Mice were administered treatments dailyby intraperitoneal injection of Compounds around the onset ofhyperglycemia (developed at about 4-8 week after birth). The threecompounds (i.e., Compound #43, Compound #C and Compound #50, deliveringidentical concentrations of selenium) was injected daily for 43-52 daysto investigate if these seleno-organic compounds have measureableeffects against hyperglycemia in the insulin-resistant mice.

It was found that treatment with all tested compounds did not affectbody weight gains in these mutant mice (data not shown), indicating thatthe tested compounds likely has little or no inhibitory effects on theabnormally increased appetite for consumption of food displayed inLepr_(db/db) mice. Also, there was no visible difference in animal grossmorphology and walking behavior between saline-treated (control) andcompound-treated Lepr^(db/db) mice during the treatment period (data notshown). These results indicate that these compounds at the tested doseshad no toxic effects on animal behavior or activity.

Among compounds #C, #50, and #43, treatment with Compounds #43 resultedin the most significant decrease, about 45% reduction compared tocontrols, of glucose levels in the bloodstream of Lepr^(db/db) mice (seeFIG. 3 left panel), even though the blood glucose levels in Compound#43-treated Lepr^(db/db) mice were still higher than normal wild-typemice of the same age (about 100 mg/dL, data not shown). Furthermore,these results clearly demonstrate that Compound #43 can significantlyreduce glucose levels in the bloodstream in this severe type II diabetesmouse model, indicating the potential of this compound for theprevention of hyperglycemia. In addition, these results provide goodevidence for differential effects of selenoorganic compounds againsthyperglycemia and show that Compound #43 is the most potent compound(among these three tested compounds) for the treatment of hyperglycemia.

In addition, sera were collected from Compound #C-, #50- and #43-treatedLepr^(db/db) mice, and subjected to an HbA1c test. HbA1c levelsrepresent a longer-term index of blood glucose concentrations over thepast 2 to 3 months, and the test is widely used in clinical medicine tomonitor blood glucose level history in diabetic patients. As shown inFIG. 3, among compounds #C, #50 and #43, Compound #43 treatmentsresulted in the only significant decrease (about a 20% reduction) inserum HbA1c levels when compared to the saline-treated group. Theseresults are consistent with the above observed differential effects ofthese three compounds on the fasting glucose levels in mice (the leftpanel in FIG. 3) and on the glucose production in vitro (HepG2 cells,FIG. 1).

Therefore, the results provide in vivo evidence that there exists adifferential effect of seleno-organic compounds against hyperglycemiaand that Compound #43 is the most potent compound among these threeselenium-containing compounds (#C, #50, and #43) with a clear potentialfor the treatment of hyperglycemia in insulin-resistant subjects.

2. Replacement of the Selenium Molecule in Compound #43 by a SulfurMolecule at the Tested Dose has No Significant Effect AgainstHyperglycemia in Lepr^(db/db) Mice after Chronic Treatments

The in vitro studies showed that Compound #43, but not Compound #68,robustly inhibits glucose production in HepG2 cells (FIG. 1). Thestudies above show that Compound #43 can lower glucose output and HbA1clevels in Lepr^(db/db) mice (FIG. 3). To further confirm theanti-diabetic effects of Compound #43 in Lepr^(db/db) mice, and toinvestigate whether the selenium molecule in Compound #43 is requiredfor this anti-hyperglycemia effect, 38-day-old male Lepr^(db/db) micewere administered treatments daily by intraperitoneal injection of equalamount of selenium or sulfur in compound #43 and its directsulfur-containing analog, Compound #68, for 3 months.

Once again, there were no visible morphological, walking behaviorabnormalities or body weight changes in Lepr^(db/db) mice aftertreatment with compounds for the 3-month period, indicating thatCompound #43 or #68 at the tested dose had no overt toxic effects onthese mice. However, injection of Compound #43 for a period of 3 monthsresulted in a statistically significant decrease in fasting bloodglucose levels, while Compound #68 did not significantly lower bloodglucose levels (FIG. 4, left panel). More dramatically, the fastingglucose levels after Compound #43 treatments for 3 months were decreasedto about 135 mg/dL, which approaches blood glucose levels reported fornormal non-diabetic/obese mice (about 100 mg/dL). In agreement withthese reduced blood glucose levels, HbA1c levels in the blood samplesfrom Lepr^(db/db) mice after Compound #43 treatment for 3 months werealso significantly decreased (by about 30%), compared to the control(saline-treated) group. However, Compound #68 treatments did not affectblood HbA1c levels in Lepr^(db/db) mice. The extent of decreased HbA1clevels in Lepr^(db/db) mice after this 90-day-treatment with Compound#43 (FIG. 4) appears to be more pronounced than in Lepr^(db/db) micefollowing 42 days of Compound #43 treatment (FIG. 3).

Together, these in vivo results further validate the finding thatCompound #43 can significantly lower blood glucose and HbA1c levels in amouse model of aggressive Type II diabetes, indicating the potential ofCompound #43 in the treatment of hyperglycemia in diabetic patients. Inaddition, the results also confirmed that the anti-diabetic potential ofCompound #43 is lost after the replacement of the selenium atom inCompound #43 with a sulfur atom.

3. Compound #43 Exhibited Higher Anti-Hyperglycemia Potential inLepr^(db/db) Mice after Chronic Treatments than Compounds #69 and #70

The in vitro studies showed that the replacement of diacetyl groups at2′,3′positions of Compound #43 with dipropanoyl groups (Compound #69) orbutanoyl groups (Compound #70) attenuate the activity of Compound #43 inthe inhibition of glucose production in HepG2 cells (FIG. 2). Thestudies above show that Compound #43 can significantly lower glucoseoutput and HbA1c levels in Lepr^(db/db) mice (FIG. 3-4). To furtherconfirm the anti-diabetic effects of Compound #43 in Lepr^(db/db) mice,and to investigate the contribution of the diacetyl groups in Compound#43 for this anti-hyperglycemia effect, 41-day-old male Lepr^(db/db)mice were administered daily treatments, by intraperitoneal injection,of equal amount in selenium of Compounds #43, #69 and #70 for 43 and 90days.

Once again, there were no visible morphological, walking behaviorabnormalities or body weight changes in Lepr^(db/db) mice aftertreatment with these three compounds for the 90 days, indicating thatCompound #43, #69 or #70, at the tested dose, had no overt toxic effectson these mice. It was found that injection of Compound #43 for 43 daysresulted in a statistically significant decrease in fasting bloodglucose levels (about a 35% decrease from about 290 mg/dL in the salinegroup to 188 mg/dL) (FIG. 5 left-hand panel). Replacement of thediacetyl groups in Compound #43 with dibutanoyl groups (Compound #70)also resulted in a decrease of fasting blood glucose levels inLepr^(db/db) mice but less significant than Compound #43 (FIG. 5left-hand panel). Replacement of diacetyl groups in Compound #43 withdipropanoyl groups (Compound #69) resulted in a slight increase infasting blood glucose levels in Lepr^(db/db) mice (FIG. 5 left-handpanel). To further validate the above observations, these Lepr^(db/db)mice were continued to be administered daily treatments, byintraperitoneal injection, of equal amount in selenium of Compounds #43,#69 and #70 for another 47 days (a total of 90 days of daily compoundtreatment), and sera were collected and subjected to blood HbA1c assays.As shown in the right-hand panel of FIG. 5, Compound #43 treatmentresulted in a significant decrease of HbA1c levels (FIG. 5, right-handpanel). Initial assessments indicated that Compound #70 treatments alsoresulted in about 50% decrease of HbA1c levels (FIG. 5, right-handpanel), whereas Compound #69 treatments did not result in a significantdecrease of HbA1c levels (FIG. 5, right-hand panel); further review ofthese data established that neither Compound #69 nor Compound #70 hadany significant effect (FIG. 5, right-hand panel). Together, theseresults suggest that Compound #43 has a great potential for thetreatment of hyperglycemia in insulin-resistant subjects. In addition,the results demonstrate that diacetyl groups at the 2′,3′ position inCompound #43 contribute to its anti-hyperglycemia function in vivo andthat extending the diacetyl groups by one or two carbon atoms might, incertain circumstances, have a negative effect on glucose homeostasis.

4. Acute Treatment of Compound #43 Resulted in a Dose-Dependent Decreaseof Blood Glucose Levels in Lepr^(db/db) Mice

The above studies demonstrated that chronic treatment with Compound #43can significantly lower blood glucose and HbA1c levels. To determine aneffective dose range and establish the duration of response to Compound#43, 8-10-week-old Lepr^(db/db) male mice were fasted overnight and theninjected intraperitoneally with saline (containing 0.2% DMSO, themaximal injected volume of Compound #43 stock solvent), 0.0054, 0.054,0.54 and 5.4 mg Compound #43 (made by diluting the stock compound withsaline)/kg body weight. Blood glucose levels in Lepr^(db/db) mice,immediately before and after the injection at 1, 2, 3, 5 and 8 hours(under fasting conditions but having free access to water), wereexamined and the resulting blood glucose levels were plotted for eachindividual animal at each time point.

Acute single injection of the above doses of Compound #43 did not causeany visible toxic effects on gross morphological and walking behavior.In saline-treated Lepr^(db/db) mice, there was only a slight decrease(about 50-70 mg/dL) in blood glucose levels during the 8 hr time periodof the test (FIG. 6), which is consistent with the fact that theseLepr^(db/db) mice display defective glucose clearance ability. However,treatment with Compound #43 at all tested doses resulted in asignificant decrease of blood glucose levels (when compared to itssaline group at each time period) except the lowest dose of Compound #43treatment at 2 or 3 hr post-injection (in which the decreased glucoselevels were close to being statistically significant) (FIG. 6). Theresults showed that Compound #43 at all tested doses was effective inreducing blood glucose levels at 1 hr after the single injection. Thisindicates that Compound #43 can reach the relevant target tissues invivo within a short time period (i.e., 1 hr) to elicit itsglucose-lowing effect. The activity of Compound #43 in reducing bloodglucose levels under fasting conditions peaked at 2 to 5 hrpost-administration, and this effect was maintained for at least another3 hr. Since the tested animals (which had been fasted overnight andcontinued to be fasted for the tested 8 hr time period) were unable tobe further fasted, the maximal effective duration of Compound #43 actionwas not determined from these experiments.

The results demonstrate that acute treatment with Compound #43 over a1000-fold concentration range significantly reduces blood glucose levelsin a widely used animal model of insulin resistance and type 2 diabetes.Compound #43 is fast-acting (≤1 hr post-treatment) and remains activefor at least 8 hours.

5. Acute Treatment of Compound #43 Attenuates Progression toHyperglycemia in Younger Lepr^(db/db) Mice

As discussed above, Lepr^(db/db) mice display elevated plasma insulinbeginning at 10 to 14 days-of-age and hyperglycemia (i.e., high bloodsugar levels) at approximately 4-8 weeks of age (Coleman D L. 1978Diabetologia 14:141-8). To test whether Compound #43 has the potentialto attenuate the development of hyperglycemia, a single dose of Compound#43 was administered via an acute injection in younger mice. In brief,6-week-old Lepr^(db/db) male mice under normal feeding conditions wereintraperitoneally injected once with saline containing 0.2% DMSO orCompound #43 at a dose of 5.4 mg/kg body weight. 24 hr after thetreatment, blood glucose levels were determined on these mice while theyhad ad-libitum access to food and water.

As shown in FIG. 7, blood glucose levels in these young Lepr^(db/db)mice at 24 hr after saline treatment were significantly increased (about20% increase), indicating that these mice are still in the process ofdeveloping hyperglycemia. In contrast, treatment of Compound #43resulted in a significant decrease (about 20%) of blood glucose levelsin Lepr^(db/db) mice (FIG. 7).

These results suggest that Compound #43 has the potential to attenuatethe development of hyperglycemia. In addition, these studies alsoindicate that the effectiveness of Compound #43 in lowering glucoseoutput likely will last at least 24 hr in these diabetic mice underfeeding conditions.

6. Enhanced Glucose Tolerance in Diabetic Lepr^(db/db) Mice afterAdministration of Compound #43

The glucose tolerance test identifies abnormalities in the way the bodyhandles glucose after a high and rapid rise of blood sugar (e.g.,usually after a meal). Insulin plays a critical role not only in theinhibition of glucose production in the liver, but also in glucoseuptake, storage and metabolism in muscle, liver, and fat cells, causinglower glucose levels in the bloodstream.

Diabetic patients have a very low glucose tolerance either due to theirinability to produce insulin or to respond to insulin efficiently tomaintain glucose homeostasis. The in vitro studies described hereinindicate that Compound #43 not only can mimic insulin but also canbypass insulin to inhibit glucose production (FIG. 1-2). Lepr^(db/db)mice are the ideal mouse Type II diabetic model to investigate the roleof Compound #43 in maintaining glucose homeostasis, considering the factthat impaired glucose tolerance and insulin-resistance are displayed inthese mutant mice. Therefore, the effect of Compound #43 and otherstructurally similar related selenium and sulfur compounds on improvedglucose tolerance in Lepr^(db/db) mice after intraperitoneal injectionof the respective compounds into mice was investigated.

These male mice, at the age of 38 days, were injected intraperitoneallywith physiological saline (containing 0.2% DMSO), Compound #C, #43, or#50 (25 μg selenium of each compound per kilogram body weight) for 43days. At the end of treatment, these mice were fasted overnight,injected with glucose (2 g/kg body weight) and blood glucose levels weremeasured at 0.25 hours (15 minutes), 0.5 hours (30 minutes), 1 hour (60minutes) and 2 hours (120 minutes) post-glucose injection. The bloodglucose levels immediately before the glucose injection (referred to asthe zero time point) were also recorded.

As shown in FIG. 8A, a significant increase in blood glucose levelsbeginning at 0.25 hours and at all the following tested time points wasobserved in saline-treated Lepr^(db/db) mice after injection of glucose.As described herein, the glucose measurement limit of the glucometeremployed for these analyses was 600 mg/dL. Thus, glucose levels inexcess of this limit were recorded as 600 mg/dL. Accordingly, certainmeasurements at the tested time point after glucose injection,particularly for the saline-treated animals, may well representunderestimations of the true blood glucose concentrations.

In Compound #C-treated mice, blood glucose levels at all tested timepoints after glucose injection remained very high, similar to thesaline-treated group (FIG. 8A). These results suggest that Compound #Cat the tested dose did not improve glucose tolerance in theseinsulin-resistant diabetic mice.

Compound #50 treatment resulted in a significant decrease of glucoselevel at the 2 hr time point after glucose injection when compared tosaline-treated group, even though there was no obvious decrease at 0.25,0.5 or 1 hr after glucose injection in FIG. 8A. These results suggestthat Compound #50 likely has some effect in improving glucose tolerancein these diabetic mice.

In Compound #43-treated Lepr^(db/db) mice, in contrast, blood glucoselevels at 0.25, 0.5 and 1 hr after glucose injection were visibly lowerthan saline-, Compound #C- or #50-treated mice (FIG. 8A). Due to themeasurement limit of the glucometer, the extent of the decrease ofglucose levels in Compound #43-treated mice relative to the othertreatments at these time points after glucose injection was likely muchmore dramatic than that shown in FIG. 8A. At 2 hours after glucoseinjection, blood glucose levels in Compound #43-treated Lepr^(db/db)mice were much lower than saline-, Compound #C- or Compound #50-treatedlittermates (see FIG. 8A), and were almost completely back topre-injection glucose levels. The decrease of glucose levels in Compound#43-treated Lepr^(db/db) mice at 2 hours after glucose injection wassignificantly different when compared to Saline- or Compound #C-treatedmice (P<0.001). These results suggest that Compound #43, but notCompound #C, at the tested dose can almost completely restore insulinaction in these insulin-resistant diabetic mice as assessed by improvedglucose tolerance, while Compound #50 may also have some beneficialeffects in the improvement of glucose clearance.

To further confirm the great potential of Compound #43 to improveglucose clearance in Lepr^(db/db) mice and to investigate whether theselenium atom in Compound #43 is required for this effect, 38-day-oldmale Lepr^(db/db) mice were administered treatments daily byintraperitoneal injection of an equal amount of selenium or sulfur inCompound #43 or #68, respectively, for 2 months. At the end oftreatment, these mice were fasted overnight and subjected to a glucosetolerance test as described above.

As shown in FIG. 8B, a significant increase in blood glucose levels wasobserved in Compound #68-treated Lepr^(db/db) mice after injection ofglucose beginning at 0.25 hours and at all the following tested timepoints. There was no obvious decrease of blood glucose levels at the 2hr time point after glucose injection (when compared to levels at 0.25,0.5 and 1 hr time periods), indicating that Compound #68 at the testeddose has little or no effects to improve glucose tolerance in theseinsulin-resistant diabetic mice.

In Compound #43-treated Lepr^(db/db) mice, blood glucose levels beforethe glucose challenge injection were significantly lower than Compound#68-treated mice (P<0.05). These results were consistent with the aboveobservation that Compound #43 is more potent than Compound #68 inreducing fasting blood glucose levels in this diabetic mouse model (FIG.4). Blood glucose levels in Compound #43-treated Lepr^(db/db) mice at0.25, 0.5 and 1 hr after glucose injection were lower than Compound#68-treated mice. At 2 hours after glucose injection, blood glucoselevels in Compound #43-treated Lepr^(db/db) mice were significantlylower than Compound #68-treated littermates (see FIG. 8B, P<0.001). Theglucose clearance curve of Compound #43-treated Lepr^(db/db) mice inthis experiment (FIG. 8B) was almost identical to the curve observed inthe first glucose tolerance test described above (FIG. 8A). Once again,due to the measurement limit of the glucometer, the decrease of glucoselevels in Compound #43-treated mice relative to Compound #68-treatedmice at these time points after glucose injection was likely much moredramatic than that shown in FIG. 8B. Regardless, the above resultsfurther confirm that Compound #43 at the tested doses dramaticallyimproves glucose tolerance, and the replacement of the selenium atom inCompound #43 with sulfur almost completely destroys its ability tofacilitate glucose clearance in these insulin-resistant diabeticLepr^(db/db) mice.

Finally, it was investigated whether the replacement of the acetylgroups at both 2′ and 3′ positions of the ribose group of Compound #43with propanoyl or butanoyl groups could improve glucose clearance inLepr^(db/db) mice. Male 41-day-old male Lepr^(db/db) mice wereadministered treatments daily by intraperitoneal injection of an equalamount of selenium in Compound #43, #69 or #70, respectively, for 43days. At the end of treatment, these mice were fasted overnight andsubjected to a glucose tolerance test as described above.

As shown in FIG. 8C, a significant increase in blood glucose levelsbeginning at 0.25 hours and at all the following tested time points wasobserved in saline-treated Lepr^(db/db) mice after injection of glucose.There was no obvious decrease of blood glucose levels before the 1 hrtime point after glucose injection (when compared to glucose levels at0.25 and 0.5 hr time periods), while glucose levels were slightlydecreased at 2 hr after glucose injection in these insulin-resistantdiabetic mice.

Compound #69 treatment resulted in a slight but non-significant decreasein glucose levels at the 2 hr time point after glucose injection, whencompared to the saline-treated group, even though there was no obviousdecrease of blood glucose levels at 0.25, 0.5 or 1 hr after glucoseinjection in FIG. 8C. These results suggest that Compound #69 may hassome effect in improving glucose tolerance in these diabetic mice.

In Compound #70-treated mice, fasting blood glucose levels beforeglucose injection were lower than the saline-treated group (FIG. 8C).After glucose injection, especially at 2 hr time point, there was aslight but non-significant decrease in blood glucose levels in Compound#70-treated mice when compared to saline-treated mice. These resultssuggest that Compound #70, at the tested dose, like Compound #69, alsolikely has some effect in improving glucose tolerance in theseinsulin-resistant diabetic mice.

In contrast, blood glucose levels at 0.25 and 0.5 hr after glucoseinjection in Compound #43-treated Lepr^(db/db) mice were visibly lowerthan saline-, Compound #69- or #70-treated mice (FIG. 8C). At 1 hourafter glucose injection, blood glucose levels in Compound #43-treatedLepr^(db/db) mice were significantly lower than saline-, Compound #69-or Compound #70-treated littermates (FIG. 8C). At 2 hours after glucoseinjection, blood glucose levels in Compound #43-treated Lepr^(db/db)mice were also much lower than Saline-, Compound #69- or Compound#70-treated mice. The decrease in glucose levels in Compound #43-treatedLepr^(db/db) mice at 2 hours after glucose injection was significantlydifferent when compared to saline-treated mice (P<0.05). Once again, dueto the measurement limit of the glucometer, the extent of the decreaseof glucose levels in Compound #43-treated mice relative to the othertreatments at each time point after glucose injection was likely muchmore dramatic than that shown in FIG. 8C. Regardless, the resultsfurther demonstrate that Compound #43 at the tested dose candramatically improve glucose tolerance in these insulin-resistantdiabetic mice. Also these results suggest that Compound #69 and #70 mayhave some beneficial effects in the improvement of glucose clearance. Incomparing the chemical structures of Compound #43 with Compound #69 and#70, it is evident that the acetyl groups at both 2′ and 3′ positions ofthe ribose group of Compound #43 are essential for optimum glucoseclearance activity, and that replacement of these diacetyl groups inCompound #43 with dipropanoyl or dibutanoyl groups significantlyattenuate its ability to facilitate glucose clearance in theseinsulin-resistant diabetic Lepr^(db/db) mice.

In summary, the above studies demonstrate that Compound #43 at thetested dose can significantly improve glucose tolerance in Lepr^(db/db)mice. The action of Compound #43 in this process is likely mediatedthrough the improvement of insulin sensitivity in the clearance ofglucose in the skeletal muscle, liver and the adipose tissues.Furthermore, while selenium is essential for the action of Compound #43its presence is not sufficient on its own to confer glucose clearanceability on a diabetic subject. The selenium atom must be presented in avery specific chemical form. This is evidenced by the lower activity ofCompound #50 and the lack of activity of Compound C; both of which arestructurally very similar to Compound #43. In addition, the acetylgroups at both 2′ and 3′ positions of the ribose group of Compound #43are also required for maintaining its activity in glucose clearance.

Example 4: Inhibition of the Expression of the Gluconeogenic Enzyme GeneG6pc in the Liver of Diabetic Leptin Receptor (Lepr) Spontaneous NullMutant Mice and in Cultured Liver Cells after Compound #43 Treatment,Together with the Potentiation of Insulin Action in the Inhibition ofG6pc Expression in Cultured Liver Cells

Liver is the main organ for producing glucose to maintain normal glucoselevels in the blood stream. Glucose-6-Phosphatase Catalytic subunit(G6pc) is an essential enzyme for gluconeogenesis in the liver. Theeffect of Compound #43 on the regulation of G6pc expression was studiedboth in vivo and in vitro.

Materials and Methods

Compounds

Compound #43, #C, #D, #E, and #50 were synthesized in the ChemistryLaboratory of Alltech, Inc. The purities of all tested compounds wereverified to be ≥99%, as determined by HPLC.

In Vivo Treatment with Compound #43, and #50 in Lepr^(db/db) Mice

Male Lepr^(db/db) mice (C57BL/6J strain, purchased from The JacksonLaboratory) at 38-days of age were intraperitoneally injected daily withphysiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #50, orCompound #43 (25 μg selenium equivalent of each compound per kilogrambody weight, diluted in the sterile physiological saline) for 52 days.After the treatment, livers were collected and subjected RNA analysis.

Cell Lines and Cell Amplification

Human hepatoma HepG2 and mouse liver AML-12 cells were purchased fromthe American Type Culture Collection (ATCC, Manassas, Va.). HepG2 cellswere amplified in Eagle's Minimum Essential Medium (EMEM) supplementedwith 10% FBS. AML-12 cells were amplified in Dulbecco's modified Eagle'smedium and Ham's F12 (DMEM/F12) media supplemented with 10% fetal bovineserum (FBS), 40 ng/ml dexamethasone (Dex, Sigma) and 1×ITS (containing0.01 mg/ml bovine insulin, 0.0055 mg/ml human transferrin, 5 ng/mlsodium selenite) solution (Sigma).

Cell Treatments for RNA Analysis

For RNA analysis of basal G6pc expression (without the presence ofdiabetic stimuli: 8-CPT/Dex), amplified AML-12 and HepG2 cells werecultured on 24-well plates (0.5-2×10⁵ cells/well) overnight in 10% FBSITS- and Dex-free DMEM/F12 media and 10% FBS EMEM media, respectively.These cells were rinsed twice with PBS to remove residual sera. Then,the PBS-washed HepG2 cells were treated without or with insulin orCompound #43 in serum-free EMEM media for 40 hr. In some experiments,the PBS-washed AML-12 cells were incubated without or with Compound #43or other selenium compounds in serum-free DMEM/F12 media for 24 hours.In other experiments, amplified AML-12 cells were pretreated without orwith Compound #43 (150 or 300 ppb) in 10% FBS but ITS/Dex-free DMEM/F12media for 24 hr. After 24 hr treatment, AML-12 cells were washed twicewith PBS (to remove any residual serum in the culture) and then treatedwith insulin, Compound #43 or both, in the serum-free DMEM/Dex media for6 hr.

For RNA analysis of the diabetic stimuli-induced G6pc expression, theAML-12 cells were pretreated without or with Compound #43 (150 or 300ppb) in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr. Then thesecells were washed twice with PBS remove any residual sera, and incubatedwith Compound #43 (150 or 300 ppb) in the presence or absence of insulin(10 or 100 nM), or 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex in serum-freeplain DMEM/F12 media for another 6 hours.

RNA Isolation and Real-Time PCR Analysis

Total RNA from saline- or selenium compound-treated Lepr^(db/db) micewas isolated using a Qiagen RNAeasy RNA isolation kit according to theManufacturer's protocol. Total RNA from cultured cells was isolatedusing Trizol (Invitrogen) according to the manufacturer's protocol, andthen incubated with DNase I to remove any potential contaminatinggenomic DNA. RNA samples were subjected to real-time PCR (QRT-PCR)analysis using Applied-Bioscience's RT kit and predesigned Taqman probes(Invitrogen), as described previously (Lan et al EMBO J 2003). Data werenormalized by Actin B (Actb) mRNA levels in each sample, and arepresented as mean±SEM of 3-5 samples.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of differences between treatment groups with aP value less than 0.05 deemed to be statistically significant.

Results:

1. Analysis of G6pc mRNA Expression in the Livers of Lepr^(db/db) Mice

Previous experiments showed different effects of Compound #43 and #50 inthe inhibition of blood glucose levels and HbA1c levels in Lepr^(db/db)mice (FIG. 3). Without wishing to be bound to any particular hypothesis,such different effects might be due, at least in part, to potentialdifferential action of these compounds on expression of thegluconeogenic G6pc gene in vivo. Therefore, the G6pc mRNA expression inLepr^(db/db) mice was measured after chronic treatment of these twocompounds.

As shown in FIG. 9, G6pc mRNA levels in the liver was slightly, but notsignificantly, decreased in Lepr^(db/db) mice after the treatment ofCompound #50. However, treatment of Compound #43 resulted in a dramaticdecrease (about 56% reduction) of G6pc mRNA levels in the livers ofLepr^(db/db) mice, when compared to saline-treated controls (FIG. 9).

Together, the results provide in vivo evidence that there may be adifferential effect of these selenium compounds in the inhibition ofG6pc expression, and that Compound #43 is a potent inhibitor of G6pcexpression in the liver of these severe Type II diabetic mice. Theseresults suggest that reduced blood glucose and HbA1c levels inLepr^(db/db) mice after Compound #43 treatment (FIG. 3) are, at least inpart, due to attenuated G6pc mRNA expression. Furthermore, because G6pcexpression is modulated in response to insulin signaling and given thatLepr^(db/db) mice are insulin-resistant, the results suggest thatCompound #43 can bypass insulin or restore insulin action to regulateG6pc expression in these diabetic mice.

2. Inhibition of G6pc mRNA Expression and Potentiation of Insulin Actionin the Inhibition of G6pc Expression in Mouse and Human Liver Cellsafter Compound #43 Treatment

The above studies revealed that Compound #43 can significantly inhibitG6pc expression in diabetic mice under insulin-resistant conditions.Cultured liver cells were used to investigate (a) whether there isdifferential effect of selenium compounds on G6pc expression, (b)whether Compound #43 has direct effects on the G6pc expression in theliver and (c) whether Compound #43 can improve insulin action in theregulation of G6pc expression. Two treatment regimes (direct compoundtreatment of liver cells under serum-free condition, and thepretreatment of liver cells with compounds in the serum-containing mediafollowed by retreatment of compounds under the serum-free conditions)were performed to examine the effect of Compound #43 on G6pc expressionin these liver cells.

First, mouse liver AML-12 cells were treated without (Control), withcompound CDE combination, Compound #C, Compound #D, Compound #50 andCompound #43 at a dose of 300 parts per billion (ppb) of selenium(equivalent to 3.8 μM of each compound) in serum-free,Insulin-Transferrin-Sodium selenite supplement (ITS) and Dexamethasone(Dex)-free media for 24 hr to investigate whether there is adifferential effect of these selenium compounds on G6pc expression. Asshown in FIG. 10A, compound CDE (300 ppb of each compound) resulted in asignificant decrease in G6pc mRNA expression. However, Compound #C, #D,or #50 at the tested dose did not significantly inhibit G6pc expressionin AML-12 cells. In contrast, treatment with Compound #43 at the samedose of selenium resulted in a robust decrease (about a 60% decrease,when compared to the Control group) of G6pc expression in AML-12 cells(FIG. 10A). The extent of decreased G6pc expression (60%) after Compound#43 treatment was more pronounced than with the Compound CDE combinationtreatment (about a 40% decrease). These results suggest that there maybe a differential effect of these seleno-organic compounds on theinhibition of G6pc expression in vitro and Compound #43 is the mostpotent compound among all tested compounds in the process. This isconsistent with the above in vivo mouse studies (FIG. 9). Since thisexperiment was performed in AML-12 cells under totally serum-freeconditions, (i.e. the absence of insulin or any other growth factors)the results suggest that the Compound #43 can mimic but bypass insulinto directly inhibit G6pc expression in AML-12 cells with a potencyhigher than the Compound CDE combination.

Next, another liver cell line, human HepG2 cells, was incubated with 100nM of insulin or 600 ppb of Compound #43 in serum-free media for 40 hrto further validate the direct inhibitory effect of Compound #43 on G6PCexpression, As shown in FIG. 10B, insulin treatment resulted in asignificant decrease of G6PC expression, indicating that the insulinsignaling is functioning in HepG2 cells. Further, G6PC mRNA levels inHepG2 cells, after the treatment of Compound #43 under totallyserum-free conditions, were significantly attenuated when compared tothe Control group (FIG. 10B). The decreased G6PC expression in HepG2cells after Compound #43 treatment is consistent with the reducedglucose production observed in FIG. 1. Thus, the results further suggestthat Compound #43 can mimic but bypass insulin to directly downregulateG6PC expression and thereby inhibit glucose production in HepG2 cells.

Finally, AML-12 cells were pretreated with Compound #43 inserum-containing but ITS/Dex-free media for 24 hr followed byretreatment of this compound in FBS/ITS/Dex-free media in the presenceor absence of insulin for 6 hr to further investigate whether Compound#43 can inhibit G6pc expression and whether there is additive orsynergistic effect between insulin and Compound #43 in thedownregulation of G6pc expression. As shown in FIG. 10C, treatment of 10nM of insulin resulted in a significant decrease (about 65%) of G6pcmRNA levels when compared to Control group (1^(st) bar in FIG. 10C).Like insulin, treatment of Compound #43 (at both 150 and 300 ppb) alsoresulted in a significant decrease of G6pc expression with the decreaselevels comparable to 10 nM insulin. Furthermore, the decrease in G6pcmRNA levels was more pronounced in AML-12 after the pretreatment withCompound #43 followed by co-treatment of both Compound #43 and insulinthan with treatment using Compound #43 or insulin alone. These resultsfurther support the above observations that Compound #43 can mimic butbypass insulin to inhibit G6pc expression in AML-12 cells. The resultsalso suggest that Compound #43 can potentiate insulin action indownregulating G6pc expression in AML-12 cells.

3. Inhibition of G6pc Expression and Improvement of Insulin Action inthe Regulation of G6pc Expression after Compound #43 Treatment in AML-12Cells Cultured Under Simulated Diabetic Conditions (Stimulated by Both8-CPT and Dex)

Cyclic AMP (8-CPT) and Dex are well known stimuli of G6pc expression andglucose production in the liver, which mimics diabetic conditions invivo. To further investigate the effects of Compound #43 on G6pcexpression, G6pc mRNA expression in AML-12 cells co-treated withcell-permeable 8-(4-chlorophenylthio) cAMP (8-CPT) and Dexamethasone(Dex) were examined. In brief, AML-12 liver cells were pretreatedwithout or with 150 ppb or 300 ppb of Compound #43 in 10% FBS butITS/Dex-free DMEM/F12 media for 24 hours. After washed with PBS twice,these cells were retreated with these selenium compounds in the presenceor absence of 10 nM or 100 nM insulin, 0.1 mM 8-CPT, and 0.5 μM Dex inserum-free media for 6 hours. After these treatments, cells werecollected and subjected to QRT-PCR analysis.

As shown in FIG. 11, AML-12 liver cells treated with 8-CPT/Dex resultedin a 41.5-fold increase in the expression of G6pc mRNA (Column #1 vs#2). Treatment with both doses of insulin significantly decreased8-CPT/Dex-induced G6pc expression in AML-12 cells, when compared to the8-CPT/Dex group (Column #3-4 vs Column #2 in FIG. 11). Further, Compound#43 at the doses of 150 and 300 ppb also significantly attenuated8-CPT/Dex-induced G6pc expression (decreased from 41.5 in Column #2 to13 in Column #5 and 13.5 in Column #8, FIG. 11) with a potencycomparable to 10 nM insulin (Column #3, FIG. 11). These studiesdemonstrated that, like insulin, Compound #43 alone at the tested dosescan inhibit 8-CPT/Dex-induced G6pc expression (about a 68% reductionwhen compared to the 8-CPT/Dex group, Column #5 and #8 vs #2 in FIG.11).

In addition, treatment of Compound #43 in combination with insulin(Column #6-7 and Column #9-10 in FIG. 11) further inhibited8-CPT/Dex-induced G6pc expression in AML-12 cells when compared to noinsulin/Compound #43 treatment (Column #2), insulin alone (Column #3-4)or Compound #43 alone (Column #5 and #8 in FIG. 11). More dramatically,G6pc mRNA levels in the treatment with Compound #43 at 150 ppb incombination with 100 nM of insulin along with 8-CPT/Dex (Column #7), andin the treatment with Compound #43 at 300 ppb in combination with 100 nMof insulin along with 8-CPT/Dex (Column #10) were robustly decreasedfrom the levels of 8-CPT/Dex treatment alone (Column #2) to the levels(Column #7, #10) close to the no-8-CPT/Dex control group (Column #1 inFIG. 11). In other words, co-treatment of Compound #43 (150 or 300 ppb)with 100 nM of insulin almost completely abolished 8-CPT/Dex-inducedG6pc expression in AML-12 cells.

Together, these results demonstrate that, like insulin, Compound #43alone at the tested doses can inhibit 8-CPT/Dex-induced G6pc expressionand the combination of both insulin and Compound #43 was even moreeffective than insulin alone or Compound #43 alone in inhibitingincreased expression of G6pc due to 8-CPT/Dex treatment in AML-12 cells.

The effects of the above decreased G6pc expression in response to theselenium compounds was not due to the potential toxic effects of theseselenium compounds on cell survival, since these compounds at the testeddose did not affect the viability of AML-12 or HepG2 cells under thesame experimental conditions (data not shown).

In summary, these results demonstrated that there is a differentialeffect of Compound #43 and #50 on the inhibition of G6pc expression inthe liver. At a minimum, the data demonstrate that Compound #43 is apotent compound to inhibit G6pc expression in the liver both in vivo andin vitro. The studies further revealed that Compound #43 can mimic butbypass insulin to directly inhibit G6pc expression in mouse and humanliver cells cultured under both normal conditions and conditionssimulating diabetes (i.e., treatment of cells with 8-CPT/Dex). Inaddition, Compound #43 can improve insulin action to inhibit G6pcexpression in AML-12 cells cultured under both normal and simulateddiabetic conditions. Together, these results provide the molecularevidence that Compound #43 can inhibit G6pc expression in the liver bothin vivo and in vitro and thus may be a valuable treatment for Type I andType II diabetics.

Example 5: Compound #43 Mimics but Bypasses Insulin to ActivatePhosphoinositide-Dependent Protein Kinase 1 (PDK1) and Protein Kinase B(AKT) Signaling to Enhance the Phosphorylation of Forkhead Box Protein01 (FOXO1) in the Liver In Vivo and In Vitro

The Forkhead transcription factor FOXO1 plays a critical role inmetabolism, gluconeogenesis and insulin sensitivity in the liver.Intracellular activity of FOXO1 is tightly regulated bypost-translational modification. In particular, phosphorylation of FOXO1excludes FOXO1 from the nucleus, thereby blocking its access to itstarget genes such as G6pc in the liver for glucose production. Ininsulin-resistant or diabetic individuals, there is no signal to excludeFOXO1 from the nucleus, so it remains present in the nucleus andstimulates the transcription of G6pc. Increased expression of G6pcdrives gluconeogenesis, leading to hyperglycemia.

As described above, the results in vivo and in vitro demonstrated thatCompound #43 can mimic but bypass insulin action to inhibit G6pcexpression and can improve insulin action in the process. Since FOXO1 isthe major signaling molecule for gluconeogenesis and insulin sensitivityin the liver and PDK1 and AKT are two major intermediate signalingmolecules upstream of FOXO1, the question as to whether Compound #43,like insulin, will target FOXO1 and its upstream signaling molecules,PDK and AKT, in Lepr^(db/db) mice, human liver HepG2 and mouse liverAML-12 cells was examined.

Materials and Methods

Compounds

Compound #43 was synthesized in the Chemistry Laboratory of Alltech,Inc. The purity of this tested compound was verified to be ≥99%, asdetermined by HPLC.

In Vivo Treatment of Compound #43 in Lepr^(db/db) Mice and Liver ProteinPreparation

Male Lepr^(db/db) mice (C57BL/6J strain, purchased from The JacksonLaboratory) at postnatal day 38 were intraperitoneally injected dailywith physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound#43 (25 μg selenium or sulfur equivalent of each compound per kilogrambody weight, diluted in sterile physiological saline) for 52 days. Afterthe treatment, livers were collected and stored at −80° C.

Frozen liver tissues were minced in sterile ice-old PBS containingcomplete proteinase and phosphatase inhibitors (Thermo-FisherScientific, Waltham, Mass.) and subjected to homogenization using atissue homogenizer (Thermo-Fisher Scientific, Waltham, Mass.). Thesetissue homogenates were diluted in Thermo-Fisher's premade RIPA buffer(1 part homogenate/2 part of RIPA buffer) containing completeproteinase/phosphatase inhibitors to extract the proteins. Proteins inthe homogenates were extracted in RIPA buffer at 4° C. overnight. Theseovernight-extracted protein lysates were centrifuged at 12000×g for 30min at 4° C., and the protein levels in the supernatant of these tissuelysates were determined using the Pierce Micro-BCA protein assay kit(Thermo Scientific-Piece Biotechnology, Rockford, Ill.) according to themanufacturer's protocol.

Cell Culture

Human hepatoma HepG2 and mouse liver AML-12 cell lines were purchasedfrom the American Type Culture Collection (ATCC, Manassas, Va.). HepG2cells were cultured in Eagle's Minimum Essential Medium (EMEM)supplemented with 10% FBS. AML-12 cells were amplified in Dulbecco'smodified Eagle's medium and Ham's F12 (DMEM/F12) media supplemented with10% fetal bovine serum (FBS), 40 ng/ml dexamethasone (Dex, Sigma) and1×ITS (containing 0.01 mg/ml bovine insulin, 0.0055 mg/ml humantransferrin, 5 ng/ml sodium selenium) solution (Sigma).

Cell Treatments for Protein Analysis

HepG2 cells were seeded on 6-well plates (7×10⁵ cells/well) and culturedin 10% FBS EMEM media for 30 hr. Then these cells were washed twice withPBS to remove residual sera, and serum-starved in plain EMEM mediaovernight. These serum-starved HepG2 cells were treated without or withCompound #43 (600 ppb) for 0 minute (right before the treatment), 30minutes, 60 minutes, 90 minutes, 24 hr, 30 hr and 40 hr.

AML-12 cells were used to investigate whether Compound #43 can regulatePdk1/Akt/Foxo1 signaling molecules in the liver cells followinginduction with diabetic stimuli. Amplified AML-12 cells were seeded on6-well (1×10⁶ cells/well) plates and cultured in 10% FBS butITS/Dex-free DMEM/F12 media for 24 hr. Then these cells were washedtwice with PBS to remove residual sera, and serum-starved in plainDMEM/F12 media overnight. These serum-starved AML12 cells were treatedwith the diabetic stimuli 8-CPT (0.1 mM) and Dex (0.5 μM) incombination, without (Control group) or with 10 nM insulin or Compound#43 (300 ppb) in serum-free plain DMEM/F12 media for 60 minutes, 90minutes, and 6 hr, respectively.

After the above described treatments, cultured HepG2 and AML-12 cellswere rinsed twice with ice-cold PBS and lysed in ice-cold RIPA buffercontaining complete proteinase and phosphatase inhibitors (Thermo-FisherScientific, Waltham, Mass.) on ice for 30 min. Cell lysates werecollected using a cell scraper and transfer pipette, and thencentrifuged at 12000×g for 30 min at 4° C. to remove the DNA pellet andobtain the protein extract. Protein levels in the supernatant of thesecell lysates were determined using the Pierce Micro-BCA protein assaykit (Thermo Scientific-Piece Biotechnology, Rockford, Ill.) according tothe manufacturer's protocol.

Western Blot Analysis

One hundred micrograms of liver tissue proteins or five micrograms oftotal proteins from control- and compound(s)-treated HepG2 or AML-12cells were subjected to SDS-PAGE gel separation and then transferred toPVDF membranes, as described previously (Reddy et al. 2008 Science).Membranes were blocked in a phosphate-buffered saline (PBS) containing5% (w/v) of bovine serum albumin (Sigma, St. Louis, Mo.) and incubatedwith specific primary antibodies followed by the incubation withHRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000dilution, Cell Signaling Inc.). All primary antibodies except Gapdh(Li-COR, Lincoln, Nebr.) were purchased from Cell Signaling Inc.Positive signals on the membrane blots were detected using theAmersham's enhanced chemiluminescence Western Blotting Prime Detectionreagents (GE Healthcare Lifescience, Pittsburgh, Pa.). Images of theseluminescence signals on the membrane blots were captured using theLI-COR Odyssey Fc Image system (Lincoln, Nebr.). The same membrane blotwas stripped and re-blotted with another antibody as described in the GEWB ECL-prime-detection protocol (GE healthcare Lifescience, Pittsburgh,Pa.). Protein band densities in the Western blots were determined usingthe NIH ImageJ software and then normalized by Gapdh or Actb/ACTB levelin each sample. Data are presented as mean±SEM of three samples per eachgroup.

Statistical Analysis

If applicable, a Student's t-test was performed to determine thestatistical difference between two groups. A P-value less than 0.05 wasconsidered significant.

Results:

1. Enhanced Phosphorylation of Pdk1, Akt and Foxo1 in the Livers ofInsulin-Resistant Lepr^(db/db) Mice after Chronic Treatment withCompound #43

The animal studies revealed that Compound #43 can reduce blood glucoseand HbA1c levels and inhibit liver G6pc expression in Lepr^(db/db) mice(FIG. 3-7, 9). The reduced fasting glucose levels and blood HbA1c levelsare at least in part attributed to the attenuated expression of thegluconeogenic G6pc gene in the livers of Lepr^(db/db) mice. G6pcexpression in the liver is controlled by the insulin signalingPdk1/Akt/Foxo1 cascade. Therefore, the application investigates whetherthe chronic treatment of Compound #43 can restore, at least to someextent, the insulin signaling (i.e., enhancing the phosphorylation ofPdk1/Akt/Foxo) in the livers of these insulin-resistant Lepr^(db/db)mice.

Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline or Compound #43 at the dose of 25 μg selenium per kilogrambody weight daily for 52 days. After the above treatments, liver tissueswere collected and subjected to Western blot analysis using specificantibodies against the insulin signaling molecules. As shown in FIG.12A, protein signals of phosphorylated-Pdk1, -Akt at Threonine 308, and-Foxo1 at Serine 256 in the livers of Compound #43-treated mice werevisibly more abundant than those in saline-treated mice. Quantitativeanalysis of these Western blots showed the protein levels of pPdk1, pAktand pFoxo1 were significantly elevated in the livers of Lepr^(db/db)mice after treatment with Compound #43 (FIG. 12B). In contrast, therewas no significant change in total Akt levels in Compound #43-treatedmice. The increased phosphorylation of Pdk1, Akt and Foxo1 stronglysuggests that the insulin downstream signaling cascade is active in thelivers of Lepr^(db/db) mice after chronic treatment with Compound #43,even though Lepr^(db/db) mice are known to be unable to respond toinsulin. In other words, the results demonstrate that Compound #43 caneither restore insulin action at least partly, bypass insulin, or both,to stimulate the phosphorylation of Pdk1/Akt/Foxo1, resulting in theattenuation of G6pc expression and glucose production in the livers ofthese insulin-resistant diabetic mice.

2. Compound #43 Mimics but Bypasses Insulin to Transiently ActivatePDK1/AKT and Subsequently Inactivate FOXO1 in Human HepG2 Cells Culturedin Serum-Free Media

To investigate whether there is an insulin-independent but insulin-likeeffect of Compound #43 in the liver to regulate the phosphorylation ofPDK1, AKT and FOXO1, serum-starved human HepG2 cells were treated withcontrol, and 600 ppb of Compound #43 in serum-free media for varioustimes, ranging from 30 min to 48 hours. Treated cells were subjected toWestern blot analysis.

As shown in FIG. 13A, there was a visibly increased protein signal forphosphorylated PDK1 in HepG2 cells after treatment with Compound #43 for30, 60 and 90 minutes, but not at longer treatment time points (after 24hr treatment). Quantitative studies showed there was a significant andtransient increase of pPDK1 in HepG2 cells after Compound #43 treatment,with the peak increase at about 60-90 minutes after compound treatment(FIG. 13B). Similarly, a significant and transient increase ofphosphorylated AKT at T308 was also observed in HepG2 cells afterCompound #43 treatment for 30, 60, 90 minutes and 24 hr, with the peakincrease at 60 and 90 minutes (FIG. 13A, C). In contrast, total AKTprotein levels in all tested time points were not significantly alteredin HepG2 cells after Compound #43 treatment (FIG. 13A, D).

The protein levels of phosphorylated FOXO1 at T24 were significantlyincreased in HepG2 cells after treatment with Compound #43 for 90minutes and longer (FIG. 13A, E). The increased FOXO1 phosphorylationwas observed later than the events of increased pPDK1 and pAKT (FIG.13A, 13E vs 13B-C). There was no significant change in total FOXO1protein levels in HepG2 cells after treatment with Compound #43 for lessthan 24 hours (FIG. 13A, 13F). However prolonged treatment of Compound#43 (30 hr or 48 hr) resulted in a slight but statistically significantdecrease of total FOXO1 proteins in HepG2 cells (FIG. 13A, F), whichcould be due to the potentially increased proteasome protein degradationof FOXO resulting from continual elevation of phosphorylated FOXO1 inHepG2 cells. Phosphorylated FOXO1 is excluded from the nucleus meaningless nuclear FOXO1 and less G6pc expression as a direct result. Also,significantly reduced glucose production (FIG. 1) and G6PC expression(FIG. 10B) were observed in Compound #43-treated HepG2 cells, as well asreduced hyperglycemia and attenuated G6pc expression in the Compound#43-treated Lepr^(db/db) mice (FIG. 3-7, 9).

Together, the above results demonstrated that Compound #43 can mimic butbypass insulin to transiently activate PDK1 and AKT, and subsequentlyinactivate FOXO1 in human liver HepG2 cells.

3. Compound #43 Mimics but Bypasses Insulin to Transiently ActivatePdk1/Akt and Subsequently Inactivate Foxo1 in AML-12 Cells CulturedUnder Simulated Diabetic Conditions (Stimulated by Both 8-CPT and Dex)

As described in the previous Examples, Compound #43 can mimic but bypassinsulin to inhibit 8-CPT/Dex-induced G6pc expression in AML-12 cells(FIG. 11). This effect could be due to the potential insulin-likeactivity of Compound #43 to inactivate Foxo1 in these mouse liver cells.Thus, the application examined the protein expression of insulinsignaling molecules in AML-12 cells, cultured under simulated diabeticconditions (stimulated with 8-CPT and Dex).

As shown in FIG. 14A, insulin treatments enhanced the phosphorylation ofPdk1, Akt and Foxo1 at 60 and 90 minutes, indicating that AML-12 cellscultured under simulated diabetic conditions were responsive to insulin.Like insulin, Compound #43 also significantly induced thephosphorylation of Pdk1, Akt and Foxo1 in these 8-CPT/Dex-treated AML-12cells after 60 minutes of compound treatment (FIG. 14A-B). At 90 minutesafter compound treatment, a significant increase in pFoxo1 proteinlevels was observed in these AML-12 cells, while the protein levels ofall other tested molecules including pPdk1, pAkt, Akt, and Foxo1 werenot significantly altered after Compound #43 treatment (FIG. 14A,C). At6 hr of Compound #43 treatment, there was still a significant increasein pFoxo1 levels, and a slight but significant decrease in total Foxo1protein levels (FIG. 14A, D). The increased pFoxo1 and slightlydecreased total Foxo1 protein levels at 6 hr treatment were alsoobserved in AML-12 cells after the treatment with 10 nM of insulin (FIG.14A, E). Once again, the slight decrease of total Foxo1 at 6 hrtreatment could be due to the targeted proteasomal protein degradationof Foxo1 proteins resulting from continuous phosphorylation of Foxo1 inthe cytosol of these AML-12 cells which had been subjected to diabeticstimuli. The enhanced Foxo1 phosphorylation after Compound #43 treatmentmay lead to decreased nuclear Foxo1 and attenuated 8-CPT/Dex-inducedG6pc expression observed in FIG. 11. Together, the results suggest thatCompound #43, like insulin, can transiently induce phosphorylation ofPdk1 and Akt and then induce Foxo1 phosphorylation in AML-12 cellscultured under simulated diabetic conditions.

In conclusion, the above in vitro and in vivo studies demonstrated thatCompound #43 can mimic but bypass insulin to transiently activatePdk/Akt and then inactive Foxo1 in the liver.

Example 6: Enhanced Glut4 (SLC2A4) Expression in the Livers ofLepr^(db/db) Mice and Cultured Liver Cells, and Enhanced Glucose Uptakein Culture Liver Cells after Compound #43 Treatment

The in vivo studies revealed that Compound #43 can lower blood glucoselevels and improve the glucose clearance in insulin-resistantLepr^(db/db) mice (FIG. 3-8). This is partially due to attenuatedglucose production in the liver but could also result from increasedglucose uptake from the bloodstream to the peripheral tissues includingthe liver. Glut4 is a critical glucose transporter and an indirect Foxo1target gene for glucose uptake in the liver, in response to systemicinsulin stimuli. Therefore, Lepr^(db/db) mice and cultured mouse liverAML-12 cells were treated with Compound #43 to test its potential effecton Glut4 expression and glucose uptake in the liver.

Materials and Methods

Compounds

Compound #43 and Compound #50 were synthesized in the ChemistryLaboratory of Alltech, Inc. The purities of all these compounds wereverified to be ≥99%, as determined by HPLC.

In Vivo Treatment of Compound #43 and #50 in Lepr^(db/db) Mice

Male Lepr^(db/db) mice (C57BL/6J strain, purchased from The JacksonLaboratory) at postnatal day 38 were intraperitoneally injected dailywith physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound#43, or Compound #50 (25 μg selenium of each compound per kilogram bodyweight, diluted in sterile physiological saline) for 52 days. Aftertreatment, livers were collected and subjected to RNA analysis.

Cell Culture

Mouse liver AML-12 cells were purchased from the American Type CultureCollection (ATCC, Manassas, Va.). These cells were amplified inDulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12) mediasupplemented with 10% fetal bovine serum (FBS), 40 ng/ml dexamethasone(Dex, Sigma) and 1×ITS (containing 0.01 mg/ml bovine insulin, 0.0055mg/ml human transferrin, 5 ng/ml sodium selenium) solution (Sigma).

Cell Treatments for RNA Analysis

For RNA analysis of basal Glut4 (Slc2a4) expression (in the absence ofdiabetic stimuli 8-CPT/Dex), amplified AML-12 cells were cultured on24-well (1×10⁵ cells/well) plates overnight in 10% FBS ITS- and Dex-freeDMEM/F12 media. These cells were washed twice with PBS to removeresidual sera and then were incubated with vehicle (0.024% DMSO) or withCompound #43 (300 ppb) in serum-free DMEM/F12 media for 24 hours.

For RNA analysis of Glut4 expression in AML-12 cells cultured undersimulated diabetic conditions, amplified AML-12 cells were cultured on24-well (2×10⁵ cells/well) plates in 10% FBS ITS- and Dex-free DMEM/F12media overnight. Then these cells were washed twice with PBS to removeany potential residual sera and then were serum-starved in plainDMEM/F12 media overnight. These serum-starved AML-12 cells were thenincubated with vehicle (0.024% DMSO) or with Compound #43 (300 ppb) inthe presence of diabetic stimuli, 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex,in serum-free plain DMEM/F12 media for 6 and 24 hours.

RNA Isolation and Real-Time PCR Analysis

Total liver RNA from saline- or Compound #43-treated Lepr^(db/db) micewas isolated using a Qiagen RNAeasy RNA isolation kit according to theManufacturer's protocol. Total RNA from cultured cells was isolatedusing Trizol (Invitrogen) according to the manufacturer's protocol, andthen incubated with DNase I to remove any potential contaminatinggenomic DNA. RNA samples were subjected to real-time PCR analysis usingApplied-Bioscience's RT kit and predesigned Taqman probes (Invitrogen),as described previously (Lan et al EMBO J 2003). Data were normalized byActin B (Actb) mRNA levels in each sample and are presented as mean±SEMof 3-5 samples.

Glucose Uptake Assay

Equal numbers of amplified AML-12 cells were seeded on 96 well plates(1.5×10⁵/well) and cultured in 10% FBS but ITS/Dex-free DMEM/F12 mediaovernight. Then these cells were washed twice with PBS (to remove anypotential residual sera), and serum-starved in plain DMEM/F12 mediaovernight. These serum-starved AML-12 cells were treated without(basal), with insulin (10 and 100 nM) or Compound #43 (150, 300 and 600ppb) in glucose/phenol red-free DMEM media at 37° C. for 1.5 hr. Aftertreatment, media was removed and cells were washed once with PBS andincubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30 min.The 2DG-treated cells were then subjected to glucose uptake measurementusing Promega's Glucose Uptake-Glo Assay kit, according to themanufacturer's protocol. Luminescent signals were recorded using aBio-Tek luminometer.

Statistical Analysis

Where applicable, a Student's-test was used to determine the statisticalsignificance of differences among different treatment groups, with a Pvalue less than 0.05 taken to represent a significant result.

Results:

1. Analysis of Glut4 mRNA Expression in the Livers of Lepr^(db/db) Miceafter Chronic Treatment with Compounds #43 and Compound #50

Male Lepr^(db/db) mice at postnatal day 38 were intraperitoneallyinjected daily with physiological saline (0.09% NaCl) containing 0.2%DMSO, Compound #43, or Compound #50 (25 μg selenium equivalent of eachcompound per kilogram body weight, diluted in sterile physiologicalsaline) for 52 days. After these compound treatments, livers werecollected and subjected to RNA analysis of Glut4 and Actb mRNA.

As shown in FIG. 15, Compound #50 treatments numerically increased Glut4mRNA levels in the livers of Lepr^(db/db) mice (when compared to thesaline-treated group). However, Compound #43 treatment elicited a largeand significant increase (5-fold; P=0.048) in Glut4 mRNA expressionlevels in the livers of Lepr^(db/db) mice (FIG. 15). These resultssuggest that there are distinct differences between these two seleniumcompounds in the stimulation of Glut4 expression in the liver and thatCompound #43 is a potent enhancer of Glut4 expression in this organ.These results also suggest that reduced blood glucose levels andimproved glucose tolerance observed in Compound #43-treated Lepr^(db/db)mice (FIG. 3-8) could be partly due to enhanced Glut4 expression,resulting in increased glucose uptake from the bloodstream into theliver of diabetic subjects.

2. Enhanced Glut4 mRNA Expression after Compound #43 Treatment in MouseLiver AML-12 Cells with or without the Stimulation of Diabetic Stimuli(8-CPT/Dex)

The above in vivo studies revealed that Compound #43 can significantlystimulate Glut4 expression in the liver of insulin-resistantLepr^(db/db) mice. This could be due to the potential systemic effect ofCompound #43 or the potential direct effect of Compound #43 on the livertissue. To test the latter scenario, cultured liver cells were used toexamine whether Compound #43 can directly regulate Glut4 expression inthe liver.

It was investigated whether Compound #43 could regulate basal Glut4expression in normal AML-12 cells (without the application of diabeticstimuli). In brief, AML-12 cells were treated with vehicle (0.024% DMSO)and Compound #43 (300 ppb) in serum-free and ITS/Dex-free media for 24hours, and subject to RNA analysis of Glut4 and Actb expression. Asshown in FIG. 16A, treatment of Compound #43 (300 ppb) resulted in asignificant increase (about 2.1-fold increase) of Glut4 mRNA expressionin AML-12 cells. Considering that this experiment was performed onAML-12 cells cultured under totally serum-free conditions, the resultssuggest that enhanced Glut4 expression after Compound #43 treatment inAML-12 cells is insulin-, serum- or any growth factor-independent.

To further test whether Compound #43 can regulate Glut4 expression,AML-12 cells which had been subjected to diabetic stimuli were used. Inbrief, AML-12 cells were serum-starved overnight, and then incubatedwith vehicle (0.024% DMSO) or with Compound #43 (300 ppb) in thepresence of diabetic stimuli, 0.1 mM 8-CPT (Sigma) and 0.5 μM Dex, inserum-free plain DMEM/F12 media for 6 and 24 hours. As shown in FIG.16B, treatment of Compound #43 for both 6 and 24 hours resulted in asignificant increase in Glut4 mRNA expression in these AML-12 cellsco-treated with the diabetic stimuli, 8-CPT and Dex. Thus, these resultsdemonstrate that Compound #43 can directly regulate Glut4 expression inAML-12 cells cultured under the simulated diabetic conditions.

3. Compound #43 Mimics but Bypasses Insulin to Enhance Glucose Uptake inMouse Liver AML-12 Cells

Enhanced Glut4 expression suggests that Compound #43 likely can mimic,yet bypass insulin to enhance glucose uptake in the liver. To test this,glucose uptake experiments were conducted on mouse liver AML-12 cells.In brief, equal numbers of AML-12 cells were seeded on 96 well plates,cultured in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr, and thenserum-starved in plain DMEM/F12 media overnight. These serum-starvedAML-12 cells were treated with insulin (10 and 100 nM) or Compound #43(150, 300 and 600 ppb) in glucose/phenol red/serum-free DMEM media at37° C. for 1.5 hr. After the treatments, cells were incubated with 1 mM2-deoxyglucose (2DG) at room temperature for 30 minutes, and thensubjected to luminescence analysis using Promega's Glucose Uptake-GloAssay kit. The detected luminescent signals represent the glucose uptakeinto the cultured cells.

As shown in FIG. 17, treatment with 10 nM of insulin did not enhance theglucose uptake. However, treatment of 100 nM of insulin resulted in asignificant increase of glucose uptake in AML-12 cells. Further,treatment of Compound #43 at all three tested doses resulted in asignificant increase of glucose uptake, and the extent of increasedglucose uptake in AML-12 cells treated with 600 ppb of Compound #43 wascomparable to 100 nM insulin (FIG. 17). Since AML-12 cells were treatedwith Compound #43 under serum-free condition, the results indicate thatlike insulin, Compound #43 can act rapidly (less than 1.5 hr) todirectly stimulate glucose uptake in the liver cells. The increasedglucose uptake in the liver could be one of the reasons why Compound #43can lower blood glucose level and improve glucose clearance in theinsulin-resistant Lepr^(db/db) mice (FIG. 3-8).

Example 7: Enhanced Expression of the Key Downstream Molecules ofInsulin Signaling, Phosphorylated Pdk1, Akt and Foxo1, in the SkeletalMuscle of Insulin-Resistant Lepr^(db/db) Mice, and the CooperativeAction of Both Insulin and Compound #43 in the Stimulation of GlucoseUptake in the Differentiated C2C12 (Skeletal Muscle) Cells

Materials and Methods

Compounds

Compound #43 was synthesized in the Chemistry Laboratory of Alltech,Inc. The purity of this tested compound was verified to be ≥99%, asdetermined by HPLC.

In Vivo Treatment of Compound #43 in Lepr^(db/db) Mice

Male Lepr^(db/db) mice (C57BL/6J strain, purchased from The JacksonLaboratory) at 38 days of age were intraperitoneally injected daily withphysiological saline (0.09% NaCl) containing 0.2% DMSO or 0.136 mg ofCompound #43 per kilogram body weight, diluted in the sterilephysiological saline) for 52 days. After the treatment, gastrocnemiusskeletal muscle samples were collected and stored at −80° C.

Skeletal Muscle Protein Preparation and Western Blot Analysis

Frozen skeletal muscles were minced in sterile ice-old PBS containingcomplete proteinase and phosphatase inhibitors (Thermo-FisherScientific, Waltham, Mass.) and subjected to homogenization using atissue homogenizer (Thermo-Fisher Scientific, Waltham, Mass.). Thesetissue homogenates were diluted in Thermo-Fisher's RIPA buffer (1 parthomogenate/2 part of RIPA buffer) containing completeproteinase/phosphatase inhibitors to extract the proteins. Proteins inthe homogenates were extracted in RIPA buffer at 4° C. overnight. Theseovernight-extracted protein/tissue lysates were centrifuged at 12000×gfor 30 min at 4° C., and the protein levels in the supernatant of thesetissue lysates were determined using the Pierce Micro-BCA protein assaykit (Thermo Scientific-Piece Biotechnology, Rockford, Ill.) according tothe manufacturer's protocol.

One hundred micrograms of skeletal muscle proteins were subjected toSDS-PAGE gel separation and then transferred to PVDF membranes, asdescribed previously (Reddy et al. 2008 Science). Membranes were blockedin a phosphate-buffered saline (PBS) containing 5% (w/v) of bovine serumalbumin (Sigma, St. Louis, Mo.) and incubated with specific primaryantibodies followed by the incubation with HRP-conjugated anti-mouse oranti-rabbit secondary antibodies (1:5000 dilution, Cell Signaling Inc.).All primary antibodies were purchased from Cell Signaling Inc, exceptthe antibodies against β-tubulin (LI-COR bioscience). Positive signalson the membrane blots were detected using Amersham's enhancedchemiluminescence Western Blotting Prime Detection reagents (GEHealthcare Lifescience, Pittsburgh, Pa.). Images of these luminescencesignals on the membrane blots were captured using the LI-COR Odyssey FcImage system (Lincoln, Nebr.). The same membrane blot was stripped andre-blotted with another antibody as described in the GE WBECL-prime-detection protocol (GE healthcare Lifescience, Pittsburgh,Pa.). Protein band densities in the Western blots were determined usingthe NIH ImageJ software and then normalized by β-tubulin level in eachsample. Data are presented as mean±SEM of 5 animal samples.

C2C12 Cell Culture, Differentiation of C2C12 Cells, and Glucose UptakeAnalysis

The mouse myoblast C2C12 cells were purchased from the American TypeCulture Collection (ATCC, Manassas, Va.). These cells were amplified inDMEM media supplemented with 10% FBS. Equal number of C2C12 cells werethen seeded on 96-well plates (5000 cells/well) and cultured in 10% FBSDMEM media at 37° C. for 5 days. Cells were replenished daily with fresh10% FBS DMEM media. At day 5 of culture, C2C12 cells were differentiatedusing 0.5% horse serum (Sigma)-containing DMEM media (differentiationmedia) continuing for 7 days with daily replacement of freshdifferentiation media, as previously described (Misu et al, CellMetabolism 12, 483-495, 2010). At day 7 post-differentiation,differentiated C2C12 cells were rinsed with PBS twice and pretreatedwithout or with 0.006% DMSO (Compound #43 solvent) or Compound #43 (300or 600 ppb) in serum-free glucose-free DMEM media overnight. Then thesecells were washed once with PBS, and then treated without (basal) orwith insulin (200 nM), Compound #43 (300 and 600 ppb), or both insulinand Compound #43 in glucose/phenol red-free DMEM media at 37° C. for 1.5hr. After treatment, media was removed and cells were washed once withPBS and incubated with 1 mM 2-deoxyglucose (2DG) at room temperature for30 min. The 2DG-treated cells were then subjected to glucose uptakeusing Promega's Glucose Uptake-Glo Assay kit, according to themanufacturer's protocol. Luminescent signals were recorded using aBio-Tek luminometer.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of differences among different treatment groupswith a P value less than 0.05.

Results:

1. Increased Phosphorylation of Insulin Downstream SignalingMolecules-Pdk1, Akt and Foxo1—in Skeletal Muscles of Insulin-ResistantLepr^(db/db) Mice after the Chronic Treatment of Compound #43

Besides liver, skeletal muscle is the other major organ critical forglucose homeostasis in response to systemic insulin. Glucose uptake inskeletal muscle plays a key role in maintaining normal glucose levels inthe bloodstream. The animal studies revealed that Compound #43 canreduce blood glucose and HbA1c levels, as well as significantlyimproving glucose tolerance in the insulin-resistant Lepr^(db/db) mice(FIG. 3-8). These effects could be due to restoration of insulinsignaling (i.e., Pdk1/Akt) in the skeletal muscle to stimulate glucoseuptake. Therefore, it was investigated whether chronic treatment ofCompound #43 could partially repair damaged insulin signaling in theskeletal muscles of these insulin-resistant Lepr^(db/db) mice.

Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline (containing 0.2% DMSO) or Compound #43 at the dose of 0.136mg of Compound #43 per kilogram body weight daily for 52 days. After theabove treatments, skeletal muscles were collected and subjected toWestern blot analysis using specific antibodies against those insulinsignaling molecules.

As shown in FIG. 18A, the protein band densities of phosphorylated Pdk1,Akt at Threonine 308, and Foxo1 at Serine 256, but not total Akt orFoxo1, were visibly higher in the skeletal muscles of Compound#43-treated mice than saline-treated mice. Quantitative analysis ofthese Western blots showed that pPdk1 protein levels were increased inthe skeletal muscles of Lepr^(db/db) mice after the treatments ofCompound #43, when compared to saline-treated mice (FIG. 18B). Theincreased levels of pPdk1 in the skeletal muscles of Compound#43-treated mice approached statistical significance (P=0.11). Further,the protein levels of two Insulin/Pdk1 downstream signaling molecules,pAkt and pFoxo1, were significantly elevated in the skeletal muscles ofLepr^(db/db) mice after treatment with Compound #43 (FIG. 18B). Incontrast, there was no significant change of total Akt and Foxo1 proteinlevels in the skeletal muscles of Compound #43-treated mice. Theincreased phosphorylation of Pdk1 and the significant increase ofphosphorylated Akt and phosphorylated Foxo1 suggest that the insulindownstream signaling cascade is active in the skeletal muscles ofLepr^(db/db) mice after the chronic treatment of Compound #43, eventhough Lepr^(db/db) mice are known to be unable to respond to insulin.In other words, the results demonstrate that Compound #43 can restoreinsulin action, bypass insulin or both, to activate PI3K to induce thephosphorylation of Pdk1/Akt in skeletal muscle, thus allowing them toperform their key functions in the regulation of glucose homeostasis inthese severely insulin-resistant type I diabetic mice.

2. Enhanced Glucose Uptake in Differentiated Mouse C2C12 (SkeletalMuscle) Cells after the Treatment of Both Insulin and Compound #43

In humans and other mammals, skeletal muscle normally accounts for 75%of whole body insulin-stimulated glucose uptake. Impaired ability ofskeletal muscle to respond to insulin is severely disruptive to systemicglucose homeostasis. It is well documented that the process of glucoseuptake in skeletal muscle is mainly mediated through the PI3K/Pdk1/Aktsignaling cascade in response to insulin. The activation ofPI3K/Pdk1/Akt signaling molecules in the skeletal muscle of theinsulin-resistant Lepr^(db/db) mice (FIG. 18) suggests that Compound #43likely can directly regulate glucose uptake in the skeletal muscle andpotentiate insulin action in the process. To test these possibilities,glucose uptake experiments were conducted on the differentiated mouseC2C12 (skeletal muscle) cells.

In brief, equal numbers of C2C12 cells were seeded on 96-well plates(5000 cells/well), and cultured in 10% FBS DMEM media at 37° C. for 5days. These cells were differentiated using 0.5% horse serum(Sigma)-containing DMEM media for 7 days to become skeletal musclecells, as previously described (Misu et al, Cell Metabolism 12, 483-495,2010). The completely differentiated C2C12 cells were pretreated withoutor with 0.006% DMSO (Compound #43 solvent) or Compound #43 (300 or 600ppb) in serum-free glucose-free DMEM media overnight. Then these cellswere incubated without (basal) or with insulin (200 nM), Compound #43(300 and 600 ppb), or both insulin and Compound #43, in glucose/phenolred-free DMEM media at 37° C. for 1.5 hr. After treatments, cells wereincubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30minutes, and then subjected to luminescence analysis using the Promega'sGlucose Uptake-Glo Assay kit. The detected luminescent signals representthe glucose uptake into the cultured differentiated C2C12 cells.

As shown in FIG. 19, treatment with 200 nM of insulin alone or 600 ppbof Compound #43, but not 300 ppb of Compound #43, resulted in a 16-19%increase (albeit not statistically significant, when compared to basalgroup) of glucose uptake in these cultured skeletal muscle cells. Sincethere was a trend towards increased glucose uptake in these culturedskeletal muscle cells after the treatment of the higher dose (600 ppb)of Compound #43 with the potency similar to 200 nM of insulin, it ispossible that Compound #43 at a dose of higher than 600 ppb can directlyand significantly enhance glucose uptake in these differentiated musclecells. Regardless, the results showed that Compound #43 at the testeddose of 600 ppb can stimulate glucose uptake with a potency comparableto 200 nM insulin.

Co-treatment of both insulin (200 nM) and Compound #43 (300 ppb)resulted in a robust and significant increase (63%) in glucose uptake bydifferentiated C2C12 cells (FIG. 19). A more pronounced increase (79%)of glucose uptake was observed in these differentiated cells after theco-treatment of 200 nM of insulin and 600 ppb of Compound #43. Theextent of increased glucose uptake in differentiated C2C12 cells afterco-treatment of both insulin and Compound #43 were much higher thaninsulin or Compound #43 alone, indicating that there was a synergisticaction between insulin and Compound #43. Therefore, the above studiesdemonstrate that Compound #43 can cooperate with insulin tosignificantly enhance glucose uptake in differentiated skeletal musclecells.

Together, the above studies suggest that Compound #43 can activate orrestore the insulin signaling (as indicated by the enhancedphosphorylation of Pdk1/Akt/Foxo1) in the skeletal muscle ofinsulin-resistant diabetic Lepr^(db/db) mice, and can potentiate insulinaction to enhance the glucose uptake in the cultured skeletal muscle(differentiated C2C12) cells. These results provide further molecularevidence that Compound #43 can lower blood glucose levels and improvethe glucose tolerance in the insulin-resistant Lepr^(db/db) mice (FIG.3-8).

Example 8: Activation of Insulin Receptor (Insr) Signaling in theSkeletal Muscle and Liver of Insulin-Resistant Lepr^(db/db) Mice and inCultured Mouse Skeletal Muscle and Human Liver Cells after Compound #43Treatment, and Insulin-Like Effects of Compound #43 on thePhosphorylation of AS160-Key for GLUT4 Translocation from CellularVesicles to Plasma Membrane for Glucose Uptake-In the DifferentiatedMouse C2C12 (Skeletal Muscle) Cells and Human Liver HepG2 Cells

It is well documented that, after the binding of insulin to the alphasubunit of Insr, Insrβ undergoes tyrosine auto-phosphorylation startingat Y1146, then at Y1150/51, which subsequently activates PI3K/Pdk1 toinduce the phosphorylation of Akt in the liver and skeletal muscle.AS160, also known as TBC1 domain family member 4 (TBC1D4), is an Aktsubstrate that plays a critical role in keeping GLUT4 proteins incytosolic vesicles. Activation of Insr/PI3k/Pdk1/Akt signaling inresponse to insulin phosphorylates AS160, promoting the translocation ofGLUT4 proteins from cytosolic vesicles to the plasma membrane tofacilitate glucose uptake in skeletal muscle cells as well as into livercells. Thus, the potential for Compound #43 to cause tyrosinephosphorylation of Insro was investigated in skeletal muscle and liverof insulin-resistant Lepr^(db/db) mice and in differentiated mouse C2C12(skeletal muscle) cells as well as human liver HepG2 cells. In addition,it was investigated whether Compound #43 could mimic but bypass insulinto activate Akt, the downstream signaling molecule of insulin receptorin differentiated mouse C2C12 cells. Further, the effects of Compound#43 on the phosphorylation of the Akt target substrate, AS160, wereinvestigated in both differentiated mouse C2C12 and human liver HepG2cells.

Materials and Methods

Compound

Compound #43 was synthesized in the Chemistry Laboratory of Alltech,Inc. The purity of this tested compound was verified to be ≥99%, asdetermined by HPLC.

In Vivo Treatment of Compound #43 in Lepr^(db/db) Mice

Male Lepr^(db/db) mice (C57BL/6J strain, purchased from The JacksonLaboratory) at postnatal day 38 were intraperitoneally injected dailywith physiological saline (0.09% NaCl) containing 0.2% DMSO, or 0.136 mgof Compound #43 per kilogram body weight, diluted in the sterilephysiological saline) for 52 days. After the treatment, gastrocnemiusskeletal muscle and liver samples were collected and stored at −80° C.

Skeletal Muscle and Liver Protein Preparation

Frozen skeletal muscle and liver tissues were minced in sterile ice-oldPBS containing complete proteinase and phosphatase inhibitors(Thermo-Fisher Scientific, Waltham, Mass.) and subjected tohomogenization using a tissue homogenizer (Thermo-Fisher Scientific,Waltham, Mass.). These tissue homogenates were diluted inThermo-Fisher's premade RIPA buffer (1 part homogenate/2 part of RIPAbuffer) containing complete proteinase/phosphatase inhibitors to extractthe proteins. Proteins in the homogenates were extracted in RIPA bufferat 4° C. overnight. These overnight-extracted protein/tissue lysateswere centrifuged at 12000×g for 30 min at 4° C., and the protein levelsin the supernatant of these tissue lysates were determined using thePierce Micro-BCA protein assay kit (Thermo Scientific-PieceBiotechnology, Rockford, Ill.) according to the manufacturer's protocol.

Cell Culture of HepG2 and C2C12 Cells, Differentiation of C2C12 Cells,Cell Treatments and Preparation of Cultured Cell Protein Extracts

The human hepatoma HepG2 cells and mouse myoblast C2C12 cell lines werepurchased from the American Type Culture Collection (ATCC, Manassas,Va.). HepG2 cells were cultured in Eagle's Minimum Essential Medium(EMEM) supplemented with 10% FBS, while C2C12 cells were amplified inDMEM media supplemented with 10% FBS.

HepG2 cells were seeded on 6-well plates (7×10⁵ cells/well) and culturedin 10% FBS EMEM media for 30 hr. Then these cells were washed twice withPBS to remove residual sera, and serum-starved in plain EMEM mediaovernight. These serum-starved HepG2 cells were treated without or withCompound #43 (600 ppb) in serum-free media for 0 minute (right beforethe treatment), 30 and 60 minutes.

To differentiate the C2C12 cells, equal number of these myoblast cellswere firstly seeded on 12-well plates (60,000 cells/well) and culturedin 10% FBS DMEM media at 37° C. for 5 days. These cells were replenisheddaily with fresh 10% FBS DMEM media. At day 5 after the culture, C2C12cells were differentiated using 0.5% horse serum (Sigma)-containing DMEMmedia (differentiation media) for 7 days with daily replacement of freshdifferentiation media, similar to previous described (Misu et al, CellMetabolism 12, 483-495, 2010). At day 7 after the differentiation,differentiated C2C12 cells were rinsed with PBS twice and incubated inserum-free DMEM media overnight. Then these serum-starved cells weretreated without (basal) or with insulin (200 nM), Compound #43 (600ppb), or both insulin and Compound #43 in serum-free DMEM media at 37°C. for 5 and 60 minutes.

After the above treatments, cultured HepG2 cells or differentiated C2C12cells were rinsed twice with ice-cold PBS and lysed in ice-cold RIPAbuffer containing complete proteinase and phosphatase inhibitors(Thermo-Fisher Scientific, Waltham, Mass.) on ice for 30 min. Celllysates were collected using a cell scraper and transfer pipette, andthen centrifuged at 12000×g for 30 min at 4° C. to remove the DNA pelletand obtain the protein extract. Protein levels in the supernatant ofthese cell lysates were determined using the Pierce Micro-BCA proteinassay kit (Thermo Scientific-Piece Biotechnology, Rockford, Ill.)according to the manufacturer's protocol.

Western Blot Analysis

One hundred micrograms of skeletal muscle or liver tissue proteins, fivemicrograms of HepG2 cell protein extracts, or eight micrograms ofdifferentiated C2C12 cell protein extracts were subjected to SDS-PAGEgel separation and then transferred to PVDF membranes, as describedpreviously (Reddy et al. 2008 Science). Membranes were blocked in aphosphate-buffered saline (PBS) containing 5% (w/v) of bovine serumalbumin (Sigma, St. Louis, Mo.) and incubated with specific primaryantibodies followed by the incubation with HRP-conjugated anti-mouse oranti-rabbit secondary antibodies (1:5000 dilution, Cell Signaling Inc.).All primary antibodies were purchased from Cell Signaling Inc, exceptthe antibodies against β-tubulin (LI-COR bioscience). Positive signalson the membrane blots were detected using the Amersham's enhancedchemiluminescence Western Blotting Prime Detection reagents (GEHealthcare Lifescience, Pittsburgh, Pa.). Images of these luminescencesignals on the membrane blots were captured using the LI-COR Odyssey FcImage system (Lincoln, Nebr.). The same membrane blot was stripped andre-blotted with another antibody as described in the GE WBECL-prime-detection protocol (GE healthcare Lifescience, Pittsburgh,Pa.). Protein band densities in the Western blots were determined usingthe NIH ImageJ software and then normalized by β-tubulin or ACTB proteinlevel in each sample. Data are presented as mean±SEM of 3-5 samples pergroup.

Enzyme-Linked Immunosorbent Assay (ELISA) of Phospho-Insrβ at Y1146 orY1150/51

Liver protein samples from Saline- or Compound #43-treated Lepr^(db/db)mice were subjected to ELISA assay using the PathScan Phospho-InsulinReceptor β (Tyr1146 or Tyr1150/1151) Sandwish ELISA kits (Cell SignalingTechnology, Danvers, Mass.) according to the Manufacturer's protocols,with the exception of incubating protein extracts with the capturingpInsrβY1146 or pInsrβY1150/1151 antibodies at 4° C. overnight (insteadof incubation at 37° C. for 2 hr as described in the protocol). Fourhundred micrograms of liver protein extracts were used for the detectionof phosphor-Insrβ at Y1146, and six hundred micrograms of liver proteinextracts for the detection of phosphor-Insrβ at Y1150/1151. Theabsorbance at 450 nm (OD450) of tested samples was recorded using aBio-tek microplater reader. The level of the internal protein control,β-tubulin, in each sample was determined by Western blot analysis using100 μg of protein extract and a specific β-tubulin monoclonal antibody,followed by quantitative analysis of β-tubulin protein band density inthe Western blot using the NIH Image J software. The OD450 of eachtested sample was then normalized by its β-tubulin protein level toobtain the level of phospho-Insrβ at Y1146 or Y150/1151.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of difference among treatment groups, with a Pvalue less than 0.05 being deemed significant.

Results:

1. Enhanced Tyrosine Phosphorylation of Insrβ in Skeletal Muscles ofInsulin-Resistant Lepr^(db/db) Mice after Chronic Treatment withCompound #43

As discussed above, skeletal muscle is absolutely essential for glucosehomeostasis in response to systemic insulin. The animal studies revealedthat Compound #43 can reduce blood glucose and HbA1c levels, and improvethe glucose tolerance in the insulin-resistant Lepr^(db/db) mice (FIG.3-8). These effects could be due to the potential restoration of insulinreceptor functioning in skeletal muscle, allowing glucose uptake tooccur in these insulin-resistant Lepr^(db/db) mice. The enhancedphosphorylation of Pdk1 and Akt in the skeletal muscle of Compound#43-treated insulin-resistant Lepr^(db/db) mice (FIG. 18) suggest thatCompound #43 may bypass but mimic insulin or restore the insulin actionto activate the insulin signaling cascade molecules upstream of thePdk1/Akt cascade in the skeletal muscle. Tyrosine phosphorylation ofInsrβ at Y1146 reflects the first step of activated insulin receptorsignaling following the binding of insulin to Insrα, and this is the keyevent upstream of PI3K/Pdk1/Akt signaling in skeletal muscle. Therefore,it was investigated whether chronic treatment with Compound #43 couldregulate the phosphorylation of Insrβ in the skeletal muscle of theseinsulin-resistant Lepr^(db/db) mice.

Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline (containing 0.2% compound solvent DMSO) or Compound #43 at adose of 0.136 mg of Compound #43 per kilogram body weight daily for 52days. After the above treatments, skeletal muscle samples were collectedand subjected to Western blot analysis using specific antibodies againstthe aforementioned insulin signaling molecules.

As shown in FIG. 20A, the protein band densities of phosphorylated Insrβat Tyrosine 1146, but not total Insrβ, were much higher in the skeletalmuscle of Compound #43-treated Lepr^(db/db) mice than saline-treatedmice. Quantitative analysis of these Western blots showed thatphosphorylated Insrβ protein levels were robustly increased (about a 2.5fold-increase) in the skeletal muscle of Lepr^(db/db) mice aftertreatment with Compound #43, when compared to saline-treated mice (FIG.20B). In contrast, there was no significant change of total Insrβprotein levels in the skeletal muscles of Compound #43-treated mice(FIG. 20C). These results are consistent with the observation ofincreased phosphorylation of Pdk1 and Akt, the key insulin signalingmolecules downstream of Insr in the skeletal muscle of Compound#43-treated Lepr^(db/db) mice (FIG. 18). Together, the results clearlydemonstrate that insulin receptor is activated in the skeletal muscle ofLepr^(db/db) mice after chronic treatment with Compound #43, even thoughLepr^(db/db) mice are engineered to be unable to respond to insulin. Inother words, the results suggest that Compound #43 can either restoreinsulin action, bypass insulin or both, to stimulate tyrosinephosphorylation of Insrβ to subsequently activate PI3K/Pdk1/Aktsignaling in the skeletal muscle of these severe type IIinsulin-resistant diabetic mice.

These findings, together with the observation of enhanced glucoseclearance from the bloodstream, reduced fasting levels of blood glucose,reduced levels of HbA1C and enhancement, in synergy with insulin, ofglucose uptake in skeletal muscle, strongly indicate that Compound #43may be an effective treatment for both type I and type II diabetes.

2. Compound #43 Mimics but Bypasses Insulin to Stimulate Phosphorylationof Insrβ at Y1146, Pdk1, Akt, and AS160 in Differentiated Mouse C2C12(Skeletal Muscle) Cells

To further investigate whether Compound #43 can directly activateinsulin receptor signaling in the skeletal muscle, serum-starveddifferentiated mouse C2C12 (skeletal muscle) cells were incubatedwithout or with Compound #43 (600 ppb), insulin (200 nM, a positivecontrol) or both in serum-free and glucose-free DMEM media for a veryshort time period (i.e., 5 minutes) and 60 minutes. Then, Western blotanalyses were performed to examine the protein expression levels ofactivated Insr (i.e., pInsrβ at Y1146) and its downstream signalingmolecules including phosphorylated Pdk1, Akt and As160 in thesedifferentiated skeletal muscle cells.

The protein expression of activated Insr in these cultured skeletalmuscle cells were measured after insulin or Compound #43 treatments. Asexpected, treatment with insulin for 5 minutes resulted in a significantincrease of pInsrβ at Y1146, but not total Insrβ, in differentiatedC2C12 cells (FIG. 21A-B), indicating that these differentiated C2C12cells rapidly respond to insulin. Further, treatment with 600 ppb ofCompound #43 also resulted in a significant increase of pInsrβ at Y1146,but not total Insrβ, in these cultured skeletal muscle cells (FIG.21A-B). Co-treatment with insulin and Compound #43 for 5 minutes tendedto increase pInsrβ protein levels in these differentiated C2C12 cells(FIG. 21A-B), indicating that the stimulation of Insrβ after Compound#43 or Insulin treatment is a transient event. Indeed, prolongedtreatments (60 minutes) with insulin, Compound #43 or both resulted in adecrease in pInsrβ levels (FIG. 21C-D), further indicating that theactivation of Insr after insulin or Compound #43 treatment is indeed atransient event and that there exists a negative feedback to regulateInsrβ tyrosine phosphorylation in skeletal muscle cells. Regardless, theenhanced tyrosine phosphorylation of Insrβ at Y1146 observed in thesecultured skeletal muscle cells after the short-time (5 minutes)treatment of Compound #43 is consistent with the activation of Insrβ inthe insulin-resistant of Lepr^(db/db) mice described above (FIG. 20).Since these differentiated C2C12 cells were serum-starved, cultured andtreated with Compound #43 under totally serum-free conditions, theresults suggest that Compound #43 can mimic but bypass insulin todirectly and quickly activate Insrβ in these differentiated skeletalmuscle cells.

Since tyrosine phosphorylation of Insrβ will activate the PI3K/Pdk1signaling to enhance the phosphorylation of Akt in response to insulin,it was investigated whether Compound #43 could mimic insulin to regulateAkt phosphorylation in these differentiated C2C12 skeletal muscle cells.As shown in FIG. 21A, treatment of insulin for 5 minutes resulted in asignificant increase of phosphorylated Akt, but not total Akt, proteinlevels. However, the increased phosphorylation of Akt was not observedin these differentiated C2C12 cells after 60 minutes of insulintreatment (FIG. 21C-D). In contrast, treatment of Compound #43 for 5minutes did not cause a significant increase of phosphorylated Aktprotein levels in these C2C12 cells, while a robust increased ofphosphorylated Akt proteins levels was observed in these cells after thetreatment of Compound #43 for 60 minutes (FIG. 21C-D). Co-treatment ofboth insulin and Compound #43 resulted in a significant increase of pAktprotein levels at both tested time points (6 min and 60 min, FIG. 21).The levels of increased phosphorylation of Akt in the insulin andCompound #43 co-treated group was comparable to insulin alone at 5minutes, while the protein levels of phosphorylated Akt at 60 minutesafter the co-treatment of both insulin and Compound #43 were nearlyidentical to the levels after the treatment of Compound #43 alone. Theseresults suggest that insulin can transiently activate the Insrdownstream signaling molecule Akt in these skeletal muscle cells.Further, the results revealed that Compound #43 could mimic but bypassinsulin to activate Akt in these cells, albeit not as quickly as insulindoes. Furthermore, the results also indicate that there was nosynergistic action between insulin and Compound #43 in the stimulationof Akt phosphorylation in these differentiated skeletal muscle cells.However, the results revealed that co-treatment of both Compound #43 andinsulin can increase the duration of the activated Akt signaling.

As discussed above, AS160 is an Akt target substrate and is required forkeeping GLUT4 proteins in the vesicles inside the skeletal muscle cells.Phosphorylation of AS160 at S588 in response to insulin/Insrβ/PI3K/Aktsignaling will cause the translocation of GLUT4 proteins from cytosolicvesicles to the plasma membrane to facilitate glucose uptake. The invivo studies revealed that Compound #43 can lower blood glucose levelsand improve glucose tolerance in the insulin-resistant Lepr^(db/db) micediabetic mice (FIG. 3-8). In addition, in cultured skeletal musclescells, increased glucose uptake was observed following Compound #43treatment, especially after co-treatment with both insulin and Compound#43 (FIG. 19). Thus it is possible that Compound #43 can regulate GLUT4translocation in skeletal muscle cells for glucose uptake through thephosphorylation of AS160. Therefore, the protein levels ofphosphorylated AS160 at S588 were measured in the differentiated C2C12(skeletal muscle) cells after the treatments of insulin, Compound #43,or both in serum-free media for 5 and 60 minutes.

As shown in FIG. 21A-B, treatment with insulin for 5 min did not affectprotein expression of AS160, but resulted in a significant increase(about 2-fold) in the phosphorylation of AS160 at S588 in thesedifferentiated C2C12 cells. However, no obvious increase inphosphorylated AS160 was observed after insulin treatment for 60 minutes(FIG. 21C-D). These results suggest that insulin can transiently enhancethe phosphorylation of AS160 for GLUT4 translocation in skeletal musclecells.

In contrast, treatment with Compound #43 alone for 5 minutes did notaffect the protein levels of phosphorylated AS160 and total AS160 inthese differentiated C2C12 cells (FIG. 21A-B). However, a robustincrease (about 6-fold) in the levels of phosphorylated AS160 at S588was observed in skeletal muscle cells after treatment with Compound #43for 60 minutes (FIG. 21C-D). These results suggest that Compound #43 canmimic insulin, albeit not as quickly as insulin does, to inducephosphorylation of AS160 for GLUT4 translocation in these culturedskeletal muscle cells.

Co-treatment with both insulin and Compound #43 at both tested timepoints (5 and 60 minutes) resulted in a significant increase ofphosphorylated AS160 protein levels (FIG. 21). The level of increasedphosphorylation of AS160 in the co-treatment group at 5 minutes wascomparable to insulin alone (FIG. 21A-B), indicating that the observedAS160 phosphorylation in the co-treatment group is mainly due to theeffect of insulin at this time point. Similarly, at 60 minutes after thetreatment, the protein levels of phosphorylated-AS160 in theco-treatment group were nearly identical to the levels after thetreatment of Compound #43 alone (FIG. 21C-D), indicating that theobserved AS160 phosphorylation in the co-treatment group at this timepoint is mainly due to the effect of Compound #43 alone. These resultssuggest that co-treatment of both Compound #43 and insulin can increasethe duration of the phosphorylation of AS160 to promote GLUT4translocation for glucose uptake in these differentiated,insulin-responsive cells.

Together, the results demonstrate that Compound #43 can mimic but bypassinsulin to directly and quickly activate Insr (indicated by tyrosinephosphorylation of Insrβ) in differentiated C2C12 cells. This isconsistent with the observed activation of Insrβ in the skeletal muscleof insulin-resistant Lepr^(db/db) diabetic mice (FIG. 20). The activatedInsr can then activate PI3K/Pdk1/Akt signaling in skeletal muscle (whichis further supported by the in vivo studies shown in FIG. 18) tosubsequently phosphorylate AS160 (a known Akt target substrate), leadingto enhanced GLUT4 translocation from cytosolic vesicles to the cellmembrane for glucose uptake. In fact, an increase of glucose uptake wasobserved in the cultured skeletal muscle cells after the treatment ofCompound #43, especially after the co-treatments of both insulin andCompound #43 (FIG. 19). Enhanced glucose uptake in skeletal muscle afterCompound #43 treatment can lower blood glucose levels and improveglucose tolerance in diabetic mice (FIG. 3-8). In short, these resultsindicate that Compound #43 can mimic but bypass insulin, to directlyactivate Insrβ/PI3K/Pdk1/Akt signaling to phosphorylate AS160 inskeletal muscle cells, resulting in enhanced GLUT4 translocation fromcytosolic vesicles to plasma membrane to promote glucose uptake intoskeletal muscle, thereby countering a key characteristic and pathologyof both type 1 and type 2 diabetes.

3. Enhanced Tyrosine Phosphorylation of Insrβ in the Liver ofInsulin-Resistant Lepr^(db/db) Mice after Chronic Treatment withCompound #43

The studies described in Example 6 revealed that Compound #43 canenhance the phosphorylation of Pdk1/Akt/Foxo1 in the liver both in vivoand in vitro (FIG. 12-14). All these effects could be attributed to theactivation of their upstream signaling molecule, Insr, in liver cellsafter Compound #43 treatment, similar to those in skeletal muscle cellsdescribed above. As discussed above, tyrosine phosphorylation of Insrβat Y1146 and, subsequently, at Y1150/1151 reflects the first few stepsof activated insulin receptor signaling following the binding of insulinto Insrα, and is the key event upstream of PI3K/Pdk1/Akt/Foxo1 signalingin the the liver. Therefore, it was investigated whether chronictreatment with Compound #43 could regulate the tyrosine phosphorylationof Insrβ in the liver of these insulin-resistant Lepr^(db/db) mice.

Lepr^(db/db) mice at postnatal day 38 were intraperitoneally injectedwith saline (containing 0.2% compound solvent DMSO) or Compound #43 atthe dose of 0.136 mg of Compound #43 per kilogram body weight daily for52 days. After the above treatments, liver samples were collected andsubjected to ELISA assays of phosphor-Insrβ at Y1146 and phosphor-Insrβat Y1150/1151 (to obtain OD450) and Western blot analysis of internalcontrol β-tubulin. The level of phosphor-Insrβ at Y1146 or at Y1150/1151in each sample was obtained after the OD450 in each sample wasnormalized by its β-tubulin protein level.

As shown in FIG. 22A, the protein levels of phosphorylated Insrβ atTyrosine 1146 were significantly increased (about a 2.9 fold-increase)in the liver of Lepr^(db/db) mice after treatment with Compound #43,when compared to saline-treated mice. Similarly, the protein levels ofphosphorylated Insrβ at Tyrosine 1150/1151 were also significantlyincreased (about a 2.95 fold-increase) in the liver of Lepr^(db/db) miceafter chronic treatment with Compound #43, when compared tosaline-treated mice (FIG. 22B). These results are consistent with theobservation of increased phosphorylation of Pdk1 and Akt, the keyinsulin signaling molecules downstream of Insr in the livers of Compound#43-treated Lepr^(db/db) mice (FIG. 12). Together, the results clearlydemonstrate that insulin receptor is activated in the liver ofLepr^(db/db) mice after chronic treatment with Compound #43, even thoughLepr^(db/db) mice are engineered to be unable to respond to insulin. Inother words, the results suggest that Compound #43 can either restoreinsulin action, bypass insulin or both, to stimulate tyrosinephosphorylation of Insrβ to subsequently activate PI3K/Pdk1/Aktsignaling in the liver of these severe type II insulin-resistantdiabetic mice.

4. Compound #43 Mimics but Bypasses Insulin to Stimulate Phosphorylationof Insrβ at Y1146 and AS160 at S588 in Human Liver HepG2 Cells

To further investigate whether Compound #43 can mimic but bypass insulinto directly activate insulin receptor in the liver cells, human liverHepG2 cells were serum-starved overnight and then were incubated withCompound #43 (600 ppb) in serum-free and glucose-free DMEM media for 30and 60 minutes. Western blot analyses were performed to examine theprotein expression levels of activated INSR (i.e., pINSRβ at Y1146) inthese human liver cells. As shown in FIG. 23, treatment with 600 ppb ofCompound #43 for both 30 and 60 minutes resulted in a significantincrease of phosphorylated INSRβ at Y1146, but not total INSRβ, in thesecultured liver cells. These results are consistent with the increasedphosphorylation of INSR downstream signaling molecules, PDK1 and AKT, inHepG2 cells after the treatment of Compound #43 for the same timeperiods (FIG. 13). Since these liver cells were serum-starved, andtreatment of Compound #43 was performed under the totally serum-freecondition, the results suggest that Compound #43 can mimic but bypassinsulin to directly stimulate tyrosine phosphorylation of INSRβ, leadingto the activation of PI3K/PI3K/AKT to inactivate FOXO1 for theinhibition of G6PC expression for glucose production, and thestimulation of the expression of GLUT4 for glucose uptake in the livercells.

Beside FOXO1, AS160 is another AKT target substrate that plays acritical role for glucose translocation from cellular vesicles to theplasma membrane in insulin-target tissues such as adipose cells andskeletal muscle. The in vivo and in vitro studies showed that Compound#43 can lower blood glucose levels and improve glucose tolerance indiabetic mice (FIG. 3-8) and can enhance glucose uptake in culturedAML-12 liver cells (FIG. 17), indicating that enhanced glucose uptake inthe liver could be one of the mechanisms of Compound #43 in loweringblood glucose levels and improving glucose tolerance against type I andII diabetes. The enhanced glucose uptake elicited after Compound #43treatment may well result from the enhanced GLUT4 expression (indicatedby FIGS. 15-16), the potential enhanced GLUT4 translocation, or both, tostimulate glucose uptake into liver cells. To address the latterscenario, phosphorylated AS160 (an AKT targeted substrate) proteinlevels were measured in Compound #43-treated HepG2 cells.

As shown in FIG. 23, treatment with 600 ppb of Compound #43 for both 30and 60 minutes resulted in a significant increase of phosphorylatedAS160 at S588, but not total AS160 protein levels, in these culturedhuman liver cells. These results are consistent with the increasedphosphorylation of PDK1 and AKT, two critical signaling moleculesupstream of AS160, in HepG2 cells after treatment with Compound #43 forthe same time periods (FIG. 13). As phosphorylation of AS160 can enhancethe GLUT4 translocation from cellular vesicles to cell membrane forglucose uptake, the results suggest that Compound #43 can mimic butbypass insulin to stimulate GLUT4 translocation from cytosolic vesicleto plasma membrane for glucose uptake in human liver cells. In supportof this, it will be recalled that enhanced glucose uptake was observedin cultured AML-12 liver cells after treatment of Compound #43 for 1.5hr (FIG. 17). Thus in liver cells, Compound #43 will not only stimulateGLUT4 expression mediated through Insr/PI3k/Pdk1/Akt/Foxo1 signaling(indicated by FIGS. 12-17), but also enhanced GLUT4 translocation fromcytosolic vesicles to plasma membrane mediated throughInsr/PI3K/Pdk1/Akt/AS160 (indicated by FIGS. 12-17, 23), thus enhancingglucose uptake.

In summary, all the above studies reveal that Compound #43 can restoreinsulin receptor function (as indicated by the enhanced tyrosinephosphorylation of Insrβ) in both skeletal muscles and liver of theinsulin-resistant diabetic Lepr^(db/db) mice. The in vitro studiesfurther demonstrate that Compound #43 can closely mimic insulin toactivate insulin receptor in both skeletal muscle cells and human livercells. In addition, the results demonstrate that Compound #43, likeinsulin, can activate Pdk1/Akt signaling to induce the phosphorylationof AS160 (an AKT target substrate) in both cultured skeletal musclecells and human liver cells. Enhanced phosphorylation of AS160 promotesGLUT4 translocation from cytosolic vesicles to plasma membrane toenhance glucose uptake in both the liver and skeletal muscle cellsresulting in lower blood glucose levels and improved glucose tolerancein diabetic situations.

It should be stressed that these two tissues, liver and skeletal muscle,are by far the most critical in the development and pathogenesis of typeII diabetes. Compound #43, through its ability to restore the insulinsignaling cascade in these tissues, could be of immense therapeuticvalue in the treatment of type II diabetes.

Furthermore, because Compound #43 can function as an insulin-mimetic ininsulin-responsive cells to which no insulin has been added, thereexists a strong possibility that it could be an effective treatment fortype I diabetes also.

Example 9: Chronic Treatments of Compound #43 Resulted in a Decrease ofSerum Insulin and Alanine Aminotransferase (ALT) Levels but not SerumCreatinine Levels in Insulin-Resistant Diabetic Db/Db Mice

Materials and Methods

Compound

Compound #43 was synthesized in the Chemistry Laboratory of Alltech,Inc. The purities of all tested compounds were verified to be ≥99%, asdetermined by HPLC.

Animals

5-week-old male diabetic spontaneous mutation (leptin receptor mutation)Lepr^(db/db) mice (C57BL/6J strain) were purchased from The JacksonLaboratory (Bar Harbor, Me.), and housed in a pathogen-free vivariumwith free access to chow and water. 3-month-old wild-type (non-diabetic)C57 mice were also purchased from the Jackson Laboratory.

Chronic Treatments with Compound #43

Male Lepr^(db/db) mice at 38 days of age were intraperitoneally (ip)injected daily with physiological saline (0.09% NaCl) containing 0.2%DMSO, and Compound #43 (0.136 mg per kilogram body weight, diluted insterile physiological saline) for 52 days. Sera from 3-month-oldwild-type (non-diabetic) C57 mice were also collected. After thetreatments, these serum samples were collected and subjected to insulin,ALT and creatinine assays.

Serum Insulin, ALT and Creatinine Assays

The serum levels of insulin, ALT and creatinine were determined usingThermo-Fisher Scientific's Insulin Mouse ELISA kit (Cat #EMINS), Sigma'sALT Activity Assay kit (Cat #MAK052), and Abcam's Creatinine Assay kit(Cat #Ab65340) according to the manufacturer's protocol, respectively.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of difference between saline- andcompound-treated groups, with a P value less than 0.05 being deemedsignificant. Data are presented as mean±SEM of the indicated numbers ofmice in the figures.

Results and Discussion

As shown in FIG. 24A, Lepr^(db/db) mice displayed hyperinsulinemia withthe insulin levels about 2586 μIU/ml (about 100 times higher thannon-diabetic wild-type mice). However compound #43 treatment resulted indramatically decrease of serum insulin levels (about 80% decrease), eventhough its level was still higher than non-diabetic wild-type mice (FIG.24A). These results suggest that compound #43 has the potential totreatment hyperinsulinemia in diabetic patients.

The alanine aminotransferase (ALT) test is typically used to detectliver injury. As shown in FIG. 24B, serum ALT levels in saline-treatedLepr^(db/db) mice were significantly higher than non-diabetic mice.However, compound #43 treatment resulted in a significant decrease ofserum ALT levels. These results suggested that the chronic treatment ofcompound #43 (treated daily for 52 days) did not display liver toxicity;instead, it may have a protective effect against liver damage.

The blood creatinine test is widely used to assess kidney function andelevated creatinine level signifies impaired kidney function or kidneydisease. As shown in FIG. 24C, there was no significant change in serumcreatinine levels in Lepr^(db/db) mice after compound #43 treatment.These results suggest that the chronic treatment of compound #43(treated daily for 52 days) likely do not have toxic effect on thekidney function.

Together the above results suggest the potential use of compound #43against hyperinsulinemia in diabetic subjects. In addition, the aboveresults also suggest that Compound #43 have little or no toxic effect onthe liver and kidney functions. Instead, Compound #43 may display someprotective effect against liver damage.

Conclusion (Mode of Action; FIG. 25)

The experimental results in this disclosure are shown by solid redarrows while the expected results (based on published literature) areshown by dashed arrows. Compound #43 can mimic but bypass insulin toquickly induce tyrosine phosphorylation of insulin receptor β subunit onthe inner surface of the cell membrane, thus precluding the need forinsulin to bind and activate insulin receptor α at the cell surface.This results in the activation of the PI3K/PDK1/AKT signaling cascade inboth liver and skeletal muscle. In other words, normal insulin signalingcan be restored without a need for insulin or active cell surfaceinsulin receptor to be present. In liver cells, activation of AKT causesa robust increase in FOXO1 phosphorylation, resulting in a significantdecrease in the expression of the FOXO1-direct target gene G6PC; thisleads to the inhibition of glucose production in the hepatocytes ofdiabetic subjects. In addition, expression of the FOXO1-indirect targetgene GLUT4 and the phosphorylation of the AKT target substrate AS160(TBC1D4), which is key for GLUT4 translocation from cytosolic vesiclesto the plasma membrane, are enhanced in liver cells after Compound #43treatment, resulting in more GLUT4 transport proteins in the liver cellmembrane and improved glucose uptake from the bloodstream. All of thispoints to improved glucose tolerance in type I and II diabetic subjects.In skeletal muscle cells, Compound #43 can also mimic, yet bypass,insulin to activate INSRβ/PDK1/AKT signaling, leading to phosphorylationof AS160 (TBC1D4). Again, this results in enhanced translocation ofGLUT4 from cytosolic vesicles to the plasma membrane to facilitateglucose uptake into skeletal muscle cells, eventually leading to asignificant decrease in blood glucose and a dramatic improvement ofglucose tolerance in both type I and II diabetic subjects. As shown,Compound #43 can potentiate insulin action by the inhibition of G6PCexpression in liver cells. Uncontrolled glucose production by liver—aprocess driven by G6PC expression—is both a key feature and a keyproblem in type 2 diabetes. Suppression of glucose production indiabetic liver is a key mechanism of action for the most widely usedclass of anti-diabetic drugs, the biguanides, e.g., metformin. Theability of compound #43 to block this process makes it potentially veryvaluable in the treatment of type 2 diabetes. Furthermore, Compound #43can restore insulin receptor function in the skeletal muscle ofinsulin-resistant diabetic mice, and can potentiate insulin action tostimulate glucose uptake in cultured skeletal muscle cells. Together,these results indicate great potential for the use of Compound #43against type I and II diabetes in humans.

Example 10: Direct Activation of Insulin Receptor Proteins by Compound#43 in a Cell-Free System

Materials and Methods

Compounds

Compound #43 and its sulfur analog, Compound #68, were synthesized inthe Chemistry Laboratory of Alltech, Inc. The purities of these testedcompounds were verified to be ≥99%, as determined by HPLC.

In Vitro Phosphorylation of Insulin Receptor (Insr), and the Detectionof Activated Insr (Indicated by Phosphorylated Tyrosine Residues at1146, 1150 and 1151 of the Insr Beta Subunit) by Western Blot Analysis

In vitro phosphorylation of Insr was performed according to Sigma'sprotocol with the following modifications. In brief, 10 μl of nativeinsulin receptor solution containing 0.8 μl of original native INSRstock solution (Sigma, Catalog #19266; diluted in enzyme dilution buffercontaining 50 mM HEPES, pH 7.6, 150 mM NaCl and 0.1% Triton X-100) wereincubated with equal volume of solutions containing insulin (Sigma),DMSO (Compound #43 solvent), Compound #43 or Compound #68 (diluted in 50mM HEPES, pH 7.6 and 100 μg/ml bovine serum albumin) on ice for 30 min.Then 20 μl of 2× kinase buffer containing 0.2 mM ATP, 50 mM HEPES, pH7.6, 50 mM MgCl₂ and 4 mM MnCl₂ were added to the above reactions, mixedand incubated on ice for 45 minutes.

Five microliters of the above reactions were immediately subjected toWestern blot analysis using specific antibodies against phosphorylatedtyrosine residues at 1146, 1150 and 1151 of Insrβ proteins (CellSignaling Inc.). Protein band density was determined using the NIH ImageJ software.

Statistical Analysis

Where applicable, a Student's t-test was used to determine thestatistical significance of difference among treatment groups, with a Pvalue less than 0.05 being deemed significant.

Results:

1. Native Insulin Receptor Protein Purified from Rat Liver Tissues wasActivated in the in Vitro Cell-Free System by Compound #43 and Insulin

Studies in cultured liver and differentiated skeletal muscle cells, aswell as in T2D diabetic mice, revealed that Compound #43 can activateinsulin receptor both in vitro and in vivo. To investigate whetherCompound #43 has a direct effect on the activation of Insr, in vitrocell-free phosphorylation assays of native insulin receptor proteins(purified from rat liver tissues) were performed. Activated Insr wasdetected using specific antibodies against phosphorylated Insrβ attyrosine residues 1146, 1150 and 1151. As expected, insulin treatmentwas able to induce the phosphorylation of Insrβ at Y1146/1150/1151, inthis cell-free in vitro system (FIG. 26). Importantly, Compound #43, atdoses of 1.9 and 3.8 μM, was also able to significantly enhance thephosphorylation of Insrβ at Y1146/1150/1151 with the elevatedphosphorylation levels being directly comparable to 0.5 μM insulin (FIG.26). These results confirm that Compound #43 can faithfully mimicinsulin to directly activate the insulin receptor, providing furthermolecular evidence that Compound #43 has the potential to replaceinsulin against diabetes, including type I diabetes.

2. Compound #68, the Sulfur Analog of Compound #43, is Far LessEffective than Compound #43 in the Activation of Insr in the Cell-FreeSystem

Studies in cultured liver cells and type 2 diabetic (T2D) Lepr^(db/db)mice showed that Compound #68, the sulfur analog of Compound #43, wasless effective than Compound #43 in the inhibition of glucose productionin vitro (FIG. 1) and the attenuation of hyperglycemia in T2D mice (FIG.4). To investigate whether there is a differential effect betweenCompound #43 and Compound #68 in the activation of Insr in the cell-freesystem, equal amounts of native insulin receptor proteins were incubatedwith the same molar concentration (3.8 μM) of Compound #43 or Compound#68, and then subjected to the in vitro phosphorylation assay. As shownin FIG. 27, Compound #43 but not Compound #68 at the tested dose wasable to activate Insr. These studies suggest that Compound #68 was lesseffective at the tested dose than Compound #43 in the activation ofInsr, which may explain the lower efficacy of Compound #68 in theinhibition of liver glucose production (FIG. 1) and againsthyperglycemia in T2D mice (FIG. 4).

Example 11: Deceased Blood Glucose Levels in Streptozotocin(STZ)-Induced Type 1 Diabetic (T1D) Mice after Acute Treatment ofCompound #43

Materials and Methods

Compounds

Streptozotocin (STZ) was purchased from Sigma. Compound #43 wassynthesized in the Chemistry Laboratory of Alltech, Inc. The purity ofCompound #43 was verified to be ≥99%, as determined by HPLC.

Type 1 Diabetic (T1D) Mouse Model and Effects of Compound #43 on BloodGlucose Levels in these T1D Mice

Five-week-old C57/BL6 male mice were intraperitoneally injected withstreptozotocin (STZ, 55 mg/kg mouse body weigh) daily for 5 days, andthen housed in the vivarium for another 14 days for recovery. Bloodglucose levels of these mice were measured using a glucometer. Thoseanimals with a blood glucose level higher than 500 mg/dL were consideredto be type I diabetic (T1D). These T1D mice with unfasted blood glucoselevels between 500-550 mg/dL were fasted overnight and injectedintraperitoneally with Compound #43 at a dose of 5.4 mg/kg body weightor physiological saline containing 2% DMSO (Compound #43 stock solvent).At 1, 2 and 3 hours post-injection, blood glucose levels in these micewere measured using the glucometer.

Statistical Analysis

A Student's t-test was used to determine the statistical significance ofdifference between control (DMSO) and Compound #43 group at each timepoint. A P value less than 0.05 denotes statistically significantdifferences between those two groups.

Results

To investigate the potential of Compound #43 against hyperglycermia inT1D mice, STZ-induced T1D mice with blood glucose levels between 500-550mg/dL were fasted overnight, intraperitoneally injected withphysiological saline containing DMSO or Compound #43 (5.4 mg/kilogrambody weight) for 1, 2 and 3 hours, and then subjected to the measurementof blood glucose levels. As shown in FIG. 28, there was no obviouschange in the blood glucose levels in control mice (with the injectionof DMSO-containing saline) during the 3-hour time period. However, atrend of decreased blood glucose levels was observed in these T1D miceafter the treatment with Compound #43 for 1 hour (FIG. 28). Moreimportantly, a significant decrease in blood glucose levels was observedin these T1D mice after Compound #43 treatment for both 2 and 3 hours(FIG. 28). Together, these results confirm that Compound #43 attenuateshyperglycemia in T1D mice.

While some embodiments are illustrated in the examples, it is apparentthat they may be altered to provide other embodiments of the instantdisclosure. Therefore, it will be appreciated that the scope of theinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of example.

The invention claimed is:
 1. A compound of formula (1):

or a pharmaceutically acceptable salt, ester, or isomer thereof, whereineach of R² and R³ is independently H or —C(O)—R, wherein each R isindependently C₁₋₆alkyl or 3-8 membered carbocyclic or heterocyclic,wherein R² and R³ cannot be both H; or R² together with R³ form—(CH₂)_(n)—C(O)—(CH₂)_(m)—, wherein each of n and m is independently0-3, and n+m≤3; R⁵ is —C₁₋₆alkyl or —C₁₋₆alkyl-CH(NH₂)COOH; R⁸ is H orhalogen; and X is H or halogen, wherein each of the carbocyclic,heterocyclic, —(CH₂)_(n)—, and —(CH₂)_(m)— moieties, independently, mayoptionally be substituted 1-3 times by —OH, halogen, NH₂, CN, orC₁₋₆alkyl; and each C₁₋₆alkyl moiety, independently, may optionally besubstituted 1-3 times by —OH, halogen, NH₂, or CN.
 2. The compound ofclaim 1, wherein R⁸ is H.
 3. The compound of claim 1, wherein X is H. 4.The compound of claim 1, wherein R⁵ is —C₁₋₆alkyl, which may optionallybe substituted 1-3 times by —OH, halogen, NH₂, or CN.
 5. The compound ofclaim 4, wherein R⁵ is unsubstituted —C₁₋₆alkyl.
 6. The compound ofclaim 5, wherein R⁵ is methyl.
 7. The compound of claim 1, wherein R⁵ is—C₁₋₆alkyl-CH(NH₂)COOH, wherein C₁₋₆alkyl may optionally be substituted1-3 times by —OH, halogen, NH₂, or CN.
 8. The compound of claim 7,wherein R⁵ is —C₁₋₆alkyl-CH(NH₂)COOH, wherein C₁₋₆alkyl isunsubstituted.
 9. The compound of claim 8, wherein R⁵ is—CH₂CH₂—CH(NH₂)COOH.
 10. The compound of claim 1, wherein R² is H, andR³ is —C(O)—R, wherein R is C₁₋₆alkyl or 3-8 membered carbocyclic orheterocyclic, wherein each of the carbocyclic and heterocyclic moieties,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl, independently, may optionallybe substituted 1-3 times by —OH, halogen, NH₂, or CN.
 11. The compoundof claim 1, wherein R³ is H, and R² is —C(O)—R, wherein R is C₁₋₆alkylor 3-8 membered carbocyclic or heterocyclic, wherein each of thecarbocyclic and heterocyclic moieties, independently, may optionally besubstituted 1-3 times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and eachC₁₋₆alkyl, independently, may optionally be substituted 1-3 times by—OH, halogen, NH₂, or CN.
 12. The compound of claim 1, wherein R is 3-8membered carbocyclic or heterocyclic, wherein each of the carbocyclicand heterocyclic moieties, independently, may optionally be substituted1-3 times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, or CN.
 13. The compound of claim 12, wherein R is 3-8 memberedunsubstituted carbocyclic or unsubstituted heterocyclic.
 14. Thecompound of claim 13, wherein R is 6 membered unsubstituted carbocyclicor unsubstituted heterocyclic.
 15. The compound of claim 14, wherein Ris unsubstituted heterocyclic.
 16. The compound of claim 15, wherein Ris


17. The compound of claim 16, wherein the compound is of formula:


18. The compound of claim 1, wherein each of R² and R³ is independentlyC(O)—R, wherein each R is independently C₁₋₆alkyl or 3-8 memberedcarbocyclic or heterocyclic, wherein each of the carbocyclic andheterocyclic moieties, independently, may optionally be substituted 1-3times by —OH, halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl,independently, may optionally be substituted 1-3 times by —OH, halogen,NH₂, or CN.
 19. The compound of claim 18, wherein each R isindependently C₁₋₆alkyl, which may optionally be substituted 1-3 timesby —OH, halogen, NH₂, or CN.
 20. The compound of claim 19, wherein eachR is independently unsubstituted C₁₋₆alkyl.
 21. The compound of claim20, wherein the compound is of the formula:


22. The compound of claim 20, wherein R is CH₃.
 23. The compound ofclaim 22, wherein the compound is of the formula:


24. The compound of claim 1, wherein R² together with R³ form—(CH₂)_(n)—C(O)—(CH₂)_(m)—, wherein each of n and m is independently0-3, and n+m≤3, wherein each of the —(CH₂)_(n)— and —(CH₂)_(m)—moieties, independently, may optionally be substituted 1-3 times by —OH,halogen, NH₂, CN, or C₁₋₆alkyl; and each C₁₋₆alkyl, independently, mayoptionally be substituted 1-3 times by —OH, halogen, NH₂, or CN.
 25. Thecompound of claim 24, wherein the —(CH₂)_(n)— and —(CH₂)_(m)— moietiesare unsubstituted.
 26. The compound of claim 24, wherein n=m=0.
 27. Thecompound of claim 26, wherein the compound is of the formula:


28. The compound of claim 1, or a pharmaceutically acceptable salt,ester, or isomer thereof, wherein the compound is of formula (2):

wherein R⁸ is H or halogen; X is H or halogen; each R₅′ is independentlyH or halogen; and each R is independently C₁₋₆alkyl, each of which,independently, may optionally be substituted 1-3 times by halogen. 29.The compound of claim 28, wherein the compound is of formula (2′):


30. The compound of claim 28, wherein R⁸ is H.
 31. The compound of claim28, wherein X is H.
 32. The compound of claim 28, wherein each R isindependently unsubstituted C₁₋₆alkyl.
 33. The compound of claim 28,wherein each R is independently C₁₋₃alkyl, each of which, independently,may optionally be substituted 1-3 times by halogen.
 34. The compound ofclaim 28, wherein each R is independently unsubstituted C₁₋₃alkyl. 35.The compound of claim 34, wherein each R is independently —CH₃, —CH₂CH₃,or —CH₂CH₂CH₃.
 36. The compound of claim 1, or a pharmaceuticallyacceptable salt, ester, or isomer thereof, wherein the compound is offormula (3):

wherein R⁸ is H or halogen; X is H or halogen; and each R′ isindependently H or halogen.
 37. The compound of claim 36, wherein thecompound is of formula (3′):


38. The compound of claim 36, wherein R⁸ is H.
 39. The compound of claim36, wherein X is H.
 40. The compound of claim 36, wherein each C(R′)₃ isindependently CF₃, CHF₂, or CH₂F, or CH₃.
 41. The compound of claim 40,wherein each C(R′)₃ is CH₃.
 42. A pharmaceutical composition comprisinga compound of claim 1, or a pharmaceutically acceptable salt, ester, orisomer thereof.
 43. The pharmaceutical composition of claim 42, whereinthe composition comprises only a single compound of formula (1), or apharmaceutically acceptable salt, ester, or isomer thereof.
 44. A methodfor treating an insulin resistance disorder comprising administering atherapeutically effective amount of a compound of claim 1, or apharmaceutically acceptable salt, ester, or isomer thereof.
 45. Themethod of claim 44, wherein the disorder is hyperglycemia, retinopathy,neuropathy, nephropathy, hyperinsulinemia, polycystic ovarian syndrome(PCOS), or a type II diabetes related vascular disorder.
 46. The methodof claim 44, wherein the step of administering comprises administering acomposition that comprises and/or delivers the compound, according to aregimen that achieves the administering of the therapeutically effectiveamount.
 47. The method of claim 46, wherein the composition is apharmaceutical composition comprising an active pharmaceuticalingredient and one or more carriers or excipients, wherein the activepharmaceutical ingredient comprises or consists of the compound offormula I.
 48. The method of claim 46, wherein the active pharmaceuticalingredient consists of the compound.
 49. A method for treating diabetescomprising administering a therapeutically effective amount of acompound of claim 1, or a pharmaceutically acceptable salt, ester, orisomer thereof.
 50. The method of claim 49, wherein the diabetes is typeI diabetes or type II diabetes.
 51. A method for treating a degenerativedisease selected from the group consisting of Alzheimer's disease,Parkinson's diseases, and sarcopenia, comprising administering atherapeutically effective amount of a compound of claim 1, or apharmaceutically acceptable salt, ester, or isomer thereof.
 52. A methodfor inhibiting glucose production, comprising administering a compoundof claim 1, or a pharmaceutically acceptable salt, ester, or isomerthereof.
 53. A method for reducing serum HbA1c level, comprisingadministering a compound of claim 1, or a pharmaceutically acceptablesalt, ester, or isomer thereof.
 54. A method for increasing glucosetolerance, comprising administering a compound of claim 1, or apharmaceutically acceptable salt, ester, or isomer thereof.
 55. A methodfor inhibiting G6pc expression, comprising administering a compound ofclaim 1, or a pharmaceutically acceptable salt, ester, or isomerthereof.
 56. A method for enhancing phosphorylation of Pdk1, Akt, AS160,and Foxo1 in the liver and/or in the skeletal muscle, comprisingadministering a compound of claim 1, or a pharmaceutically acceptablesalt, ester, or isomer thereof.
 57. A method for increasing Glut4expression, comprising administering a compound of claim 1, or apharmaceutically acceptable salt, ester, or isomer thereof.
 58. A methodfor activating and/or restoring insulin signaling in a subject ininsulin-resistant state, comprising administering a compound of claim 1,or a pharmaceutically acceptable salt, ester, or isomer thereof.
 59. Themethod of claim 58, wherein the subject is characterized by significantlevels of circulating insulin.
 60. The method of claim 58, wherein theinsulin-resistant state is characterized by a reduction in correctly orappropriate phosphorylated insulin receptor in the subject.
 61. Themethod of claim 58, wherein the subject has diabetes, and/or diabetesassociated disease, disorders, or conditions.
 62. A method for enhancingglucose uptake into cells in a subject, comprising administering acompound of claim 1, or a pharmaceutically acceptable salt, ester, orisomer thereof.
 63. The method of claim 62, wherein the cells areskeletal muscle cells and liver cells.
 64. A method for enhancingtranslocation of glucose transporter proteins (GLUTs) from cytosolicvesicles to plasma membrane for glucose uptake, comprising administeringa compound of claim 1, or a pharmaceutically acceptable salt, ester, orisomer thereof.